MOLECULAR MORPHOLOGY in HUMAN TISSUES: Techniques and Applications
Advances in Pathology, Microscopy, & Molecular Morphology Series Editors Jiang Gu and Gerhard W. Hacker PUBLISHED TITLES Gold and Silver Staining: Techniques in Molecular Morphology Gerhard W. Hacker and Jiang Gu Molecular Morphology in Human Tissues: Techniques and Applications Gerhard W. Hacker and Raymond R. Tubbs
Advances in Pathology, Microscopy & Molecular Morphology Series Editors Jiang Gu and Gerhard W. Hacker
MOLECULAR MORPHOLOGY in HUMAN TISSUES: Techniques and Applications Edited by
GERHARD W. HACKER Forschungsinstitut für Grund- und Grenzfragen der Medizin und Biotechnologie St. Johanns Spital, Landeskliniken Salzburg Salzburg, Austria, EU
RAYMOND R. TUBBS
Section Head for Molecular Genetic Pathology Professor and Chairman, Department of Clinical Pathology The Cleveland Clinic Lerner College of Medicine of Case Western Reserve University Cleveland, Ohio, USA
CRC PR E S S Boca Raton London New York Washington, D.C.
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Library of Congress Cataloging-in-Publication Data Molecular morphology in human tissues : techniques and applications / edited by Gerhard W. Hacker, Raymond R. Tubbs. p. ; cm. -- (Advances in pathology, microscopy & molecular morphology) Includes bibliographical references and index. ISBN 0-8493-1702-9 (alk. paper) 1. Histochemistry--Laboratory manuals. I. Hacker, Gerhard W. II. Tubbs, Raymond R., 1946- III. Series. [DNLM: 1. Histocytological Preparation Techniques. 2. Immunohistochemistry--methods. QW 525 M7506 2004] QH613.M65 2004 611′.018--dc22 2004050318 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. All rights reserved. Authorization to photocopy items for internal or personal use, or the personal or internal use of specific clients, may be granted by CRC Press, provided that $1.50 per page photocopied is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA The fee code for users of the Transactional Reporting Service is ISBN 0-8493-1702-9/05/$0.00+$1.50. The fee is subject to change without notice. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. The consent of CRC Press does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press for such copying. Direct all inquiries to CRC Press, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe.
Visit the CRC Press Web site at www.crcpress.com © 2005 by CRC Press No claim to original U.S. Government works International Standard Book Number 0-8493-1702-9 Library of Congress Card Number 2004050318 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper
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DEDICATION This book is dedicated to HR Dr. Alois Grüner, Director of the Health Department of “Land Salzburg” (Salzburg State) and Chairman of the Ethics Commission for Medical Research in Salzburg, Austria. I would like to thank him for nearly two decades of fruitful collaboration and encouragement in the field of frontier questions in medicine and biology. Dr. Grüner has provided outstanding contributions to the benefit of humanity in Austria, constantly working toward increasing awareness, understanding, and peace. G.W.H.
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FOREWORD Molecular advances in the biomedical sciences have been moving at a very rapid pace during the past few decades. With the completion of the sequencing of the entire human genome, these molecular advances are being used to address many questions about morphology-based methods at the tissue and cellular levels. Molecular morphology is finally coming of age. Professors Gerhard W. Hacker and Raymond R. Tubbs have produced an interesting volume that focuses on molecular morphology at the light microscope level, with an exciting combination of morphological techniques and molecular biologic methods. Many of the authors of the 18 chapters are pioneers who have made seminal contributions in their respective fields. This volume, which is the second in a series on “Advances of Pathology, Microscopy, and Molecular Morphology,” is especially attractive because it can serve as a practical laboratory and protocol book of state-of-the-art techniques in molecular morphology. The emphasis of the chapters on human tissue materials and cell culture provides a practical focus for investigators interested in many different aspects of molecular morphologic techniques. This volume should find an important place on the bookshelves of clinical investigators as well as basic scientists who are interested in analyzing molecular alterations in cells and tissues.
Ricardo V. Lloyd, M.D., Ph.D. Vernon F. and Earline D. Dale Professor Mayo Foundation Rochester, Minnesota
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PREFACE This is the second issue in the CRC Press book series entitled “Advances in Pathology, Microscopy, and Molecular Morphology.” The book is dedicated to the presentation of the most promising and modern techniques of molecular morphology. It is a comprehensive guide and a practical laboratory protocols book; each technique is described and discussed by first-class authors, each of whom enjoys a worldwide reputation in the discipline of molecular morphology. Molecular morphology is a field of microscopy, histology, and cytology that has emerged from the combination and adaptation of techniques formerly developed for immunology, biochemistry, and molecular biology. During the past decade, this field has grown enormously and evolved into a new discipline of scientific technologies and applications, useful for various areas of biomedical research and clinical diagnostics. Molecular morphology allows the microscopic visualization of biochemical/molecular biological/physiologic processes in human, animal, and plant tissues, and the useful areas include diagnostic molecular pathology, histo- and cytogenetics, and manifold other fields of medicine and biology. From a purely analytical perspective, molecular and immunological techniques have now been adapted to in situ localization, thus allowing the precise and highly specific localization of substances such as peptides, proteins, DNA, and RNA, each readily visible via conventional or adapted light and electron microscopy. The molecular pathologist today relies to a large degree on techniques of molecular morphology, thereby analyzing the location and possible alterations of substances, such as nucleic acid sequence amplification or deletion, detection of intrinsic or external genes, or immunological tumor markers demonstrated within cellular and subcellular structures. Use of these molecular tools makes the diagnosis and management of cancer, viral infections, and other diseases more precise and reliable. Furthermore, combining techniques of molecular morphology with the capabilities of modern computer imaging and processing has provided a third dimension now accessible in 3D microscopy. The latter application facilitates both the full-color demonstration of spatial structure and much more precise measurements within the images obtained in all three dimensions. The neurobiologist, for example, can apply ultrasensitive and specified amplified immunohistochemical methodologies to analyze (co)-localization and structural relationships of neuropeptide-containing terminal nerve fibers to a much more sensitive and more realistic degree than previously attainable. Applications and new technologies are being developed and modified continuously. This book distinguishes itself from other books not only by the quality of contributors included, but also in the approach to the topic: it is a type of “cookbook” of latest technology. The most robust recent developments in the discipline have been assembled and presented in a very useful, practical way, not only for experienced researchers, but also for neophytes who want to carry out productive science by relying on state-of-the-art technology. Such techniques are usually presented singly in journals, and not often in reproducible format. Very often, authors “do not tell their secrets.” In this book, they do, and they explain in detail firsthand how their techniques work and how experiments can be brought to a successful conclusion.
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Each of the 18 chapters follows a special format, similar to the first book of this series, entitled Gold and Silver Staining: Techniques in Molecular Morphology. After an introduction and a complete and accurate listing of materials (with sources of each special reagent, Web sites, and other necessary data), detailed step-by-step protocols are given, followed by a description of how the results should look. A technical hints and discussion section, key references, and figures illustrating the appropriate outcome complete each chapter. In Chapter 1, Guida Portela-Gomes, a highly reputed specialist for co-localization studies in neuroendocrinology, gives expert guidance for the practical use and application of double and multiple immunostaining techniques in peptide research and diagnosis. Chris van der Loos, the authority in multiple staining in biomedical research, provides in Chapter 2 a treasure source for everyone attempting successful combinations of various methodologies of immunohistochemistry. Recent advances in enzyme-based amplification methods, novel fluorophores, and improved detection protocols have dramatically increased the utility of fluorescence localization procedures in human tissue sections. Kevin Roth and Denis Baskin, both among the most respected authorities in histochemistry and cytochemistry, provide in Chapter 3 detailed protocols for enzyme-enhanced immunohistochemical and in situ hybridization detection and discuss practical solutions to common problems encountered using these sensitive methods. In Chapter 4, James Hainfeld and Richard Powell, the original inventors of nanogold particles and gold enhancement, provide a detailed overview of applications of clustered gold in molecular morphology. Chapter 5 is a technological highlight contributed by Raymond Tubbs and collaborators, describing a new versatile and reliable detection method for HER2 gene amplification facilitating the detection of certain cancer gene alterations in daily clinicalpathological diagnosis. Chapter 6 is dedicated to high-power signal amplification methods, by which single gene molecular detection sensitivity has been achieved. This text is contributed by the editors of this book, Gerhard Hacker and Raymond Tubbs. Microarray-based genomic hybridization as a tool for the survey of genomic alterations in human neoplasms is described in Chapter 7 by Dina Kandil and colleagues of the Cleveland Clinic. Shin-ichi Izumi and collaborators in Chapter 8 introduce their own technique, termed “southwestern histochemistry.” The senior author of this chapter is Paul Nakane, the original inventor of the immunoperoxidase method and undoubtedly one of the world’s top authorities in the field of molecular morphology. Automated mRNA in situ hybridization and tissue microarrays are described in Chapter 9 by Hiro Nitta and colleagues. The methodology described here illustrates the utility of fully automated messenger RNA detection in large numbers of different tissues on a single tissue microarray slide, and therefore can be understood as an important contribution toward increased reproducibility, standardization, and economy. Chapter 10 concerns a new method of whole mount in situ hybridization, contributed by Sabine Tontsch and colleagues. In Chapter 11, Marek Skacel and collaborators open new doors to molecular cytopathology, outlining the methodology of interphase fluorescence in situ hybridization performed on liquid-based thin-layer cytopathologic preparations. Lisa Bobroski and Omar Bagasra in Chapter 12 overview the latest developments in in situ PCR and give detailed descriptions for successfully performing this technique. Omar Bagasra is the original inventor of in situ PCR.
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Andreas Aschoff and Gustav Jirikowski in Chapter 13 describe their technique for the visualization of apoptotic markers in human biopsies in high resolution. In Chapter 14, Gerhard Hacker and co-authors introduce the 3D (three-dimensional) digital optical microscope technology. Numerous full-color 3D photomicrographs demonstrate applications of this method for molecular morphology and also give examples from general biology. Chapter 15, by Michelle Lennartz and colleagues, is dedicated to the visualization of signal transduction pathways in real time. Masahiko Zuka in Chapter 16 demonstrates his promising method to estimate the proteolytic activity by film in situ zymography. Chapter 17 is dedicated to a more ethically conscious production and purchase of reagents for molecular morphology. The medical biologist Gerhard Hacker, the biochemist Günter Schwamberger, and the attorney-at-law Antoine Goetschel in this chapter merge their three different special areas of expertise to articulate a humane viewpoint of reagent selection for molecular morphology. Last but not least, in Chapter 18, authored by Anthony Rhodes, this European pioneer of standardization and quality management in diagnostic immunocytochemistry reminds molecular morphologists of the preeminent importance of quality assurance when applying molecular techniques in diagnosis and research. The contents of this book are intended to provide reproducible, practical information on the latest technology and offer a balanced view. From the beginning, we have emphasized the use of human tissues or cell-culture-derived preparations. With this, we would like to follow a direction already given in the first book of this series, emphasizing the avoidance of animal experiments whenever possible. Again, most contributors responded to our request to do so, and we would like to thank them for their enlightened contributions. We would like to thank the distinguished authors of this book for sharing their experience with the readers. It is our intent that this publication serve as a valuable source of practical information in molecular morphology, and that it will be used in laboratories for many years to come. Gerhard W. Hacker Raymond R. Tubbs
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THE EDITORS Gerhard W. Hacker, Ph.D., Director of the Research Institute for Frontier Questions of Medicine and Biotechnology at the St. Johanns-Hospital in Salzburg, Austria, is full professor of histochemistry, histology, and endocrinology at the University of Salzburg. He obtained his Ph.D. at the Faculty of Natural Sciences at Salzburg University, and studied scientific medicine at the University of London with Prof. Dame Julia M. Polak, where he received the Diploma in Endocrinology and Pathology of the Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K. After further postdoctoral training and research periods in several institutions in Sweden and the United States, he was appointed to develop diagnostics and research laboratories in the fields of biomedicine, biotechnology, and histotechnology. His main research areas and interests include the development of improved laboratory technologies, such as immunogold-silver staining, in situ single virus copy detection (in situ PCR), supersensitive in situ hybridization, neuropeptide research, neuroendocrine tumors, stress management, medical ethics, palliative care, and animal welfare. His institute can be reached at the following Web site: http://www.frontierquestions.com. Raymond R. Tubbs, D.O., is Director of the Molecular Genetic Pathology Laboratory, Section Head for Molecular Genetic Pathology, and Chairman of the Department of Clinical Pathology at the Cleveland Clinic Foundation in Cleveland, Ohio (http://www.ccf.org). Dr. Tubbs is Professor of Pathology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University. He obtained his D.O. degree at the Kirskville College of Osteopathic Medicine, completed a medical internship at the Cleveland Clinic Foundation, a combined Anatomic and Clinical Pathology Residency at the Cleveland Clinic Foundation, and served as Clinical Associate Fellow in Immunopathology with Dr. Sharad Deodhar. He was granted a Leave for Professional Development by the Cleveland Clinic Foundation to study molecular morphology with Dr. Gerhard Hacker of the Institute of Pathological Anatomy, Salzburg. Dr. Tubbs is President of the International Society of Analytical and Molecular Morphology. His principal research areas include molecular hematopathology and the molecular pathology of solid tumors.
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ACKNOWLEDGMENTS Dr. Hacker would like to express his deep thanks to his wife, Mrs. Ursula DemarmelsHacker, for her brilliant assistance in editing this book, and for her enormous patience and understanding during the months (including countless evenings and weekends) necessary for preparation of this book. He also gratefully acknowledges the expert help of Mrs. Petra Ablinger and Dr. Ilse Jekel (both of Salzburg, Austria) during the text and photomicrograph pre-editing process. The book would not have been possible without the friendly support of the directory board of the St. Johanns-Hospital of the Salzburger Landeskliniken, especially by Prof. Gernot Pauser, Medical Director. Dr. Tubbs would like to express his deep thanks to his wife and best friend of 35 years, Mary Tubbs, for her faithful and loving support. He also gratefully acknowledges the outstanding scientific expertise and dedication of Jim Pettay, MT (ASCP), Lead Research Technologist and Supervisor of the Molecular Genetic Pathology Laboratory of the Cleveland Clinic, and the expert help of Mrs. Willa Dise in the preparation of the text.
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CONTRIBUTORS Isabella Artner Department of Molecular Physiology and Biophysics Vanderbilt University Medical Center Nashville, Tennessee Andreas P. Aschoff Institut für Anatomie II, FSU Jena Jena, Germany Omar Bagasra Professor Department of Biology Claflin University Orangeburg, South Carolina Denis G. Baskin Professor Departments of Medicine (Division of Metabolism, Endocrinology and Nutrition) and Biological Structure University of Washington and VA Puget Sound Health Care System Seattle, Washington Hans-Christian Bauer Professor Department of Organismal Biology University of Salzburg Salzburg, Austria Lisa Bobroski Windber Research Institute Windber, Pennsylvania Pamela M. Brannock Center for Cell Biology and Cancer Research Albany Medical College Albany, New York Erinn Downs-Kelly Department of Anatomic and Clinical Pathology The Cleveland Clinic Foundation Cleveland, Ohio
Antoine F. Goetschel Director and Attorney-at-Law Stiftung für das Tier im Recht (Foundation for the Animal in the Law) Zurich, Switzerland Thomas Grogan Professor of Pathology University of Arizona Health Sciences Center, Tucson, Arizona and Chief Medical Officer Ventana Medical Systems, Inc. Tuscon, Arizona Gerhard W. Hacker Professor and Director Research Institute for Frontier Questions of Medicine and Biotechnology Salzburger Landeskliniken, St. Johanns Hospital Salzburg, Austria James F. Hainfeld Department of Biology Brookhaven National Laboratory Upton, New York Marybeth Hartke Department of Clinical Pathology The Cleveland Clinic Foundation Cleveland, Ohio David G. Hicks Section Head, Surgical Pathology Department of Anatomic Pathology The Cleveland Clinic Foundation Cleveland, Ohio Shin-ichi Izumi Instructor Division of Oral Cytology and Cell Biology Nagasaki University Graduate School of Biomedical Sciences Nagasaki, Japan xvii
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Gustav F. Jirikowski Professor and Director Institut für Anatomie II, FSU-Jena Jena, Germany Dina Kandil Molecular Pathology Research Fellow Department of Clinical Pathology The Cleveland Clinic Foundation Cleveland, Ohio Takehiko Koji Professor Division of Histology and Cell Biology Nagasaki University Graduate School of Biomedical Sciences Nagasaki, Japan Michelle R. Lennartz Associate Professor Center for Cell Biology and Cancer Research Albany Medical College Albany, New York Günter Lepperdinger Institut für Biomedizinische Alternsforschung Österreichische Akademie der Wissenschaften Innsbruck, Austria Joseph E. Mazurkiewicz Professor Center for Neuropharmacology and Neuroscience and Director AMC Imaging Core Facility Albany Medical College Albany, New York Paul K. Nakane Professor Environmental Biotechnology Research Institute Biological Sciences Department California Polytechnic State University San Luis Obispo, California Hiro Nitta Ventana Medical Systems, Inc. Tucson, Arizona xviii
James D. Pettay Lead Research Technologist and Supervisor Molecular Genetic Pathology Laboratory Department of Clinical Pathology The Cleveland Clinic Foundation Cleveland, Ohio Guida M. Portela-Gomes Associate Professor in Experimental Pathology Center of Nutrition and Metabolism University of Lisbon Lisbon, Portugal Richard D. Powell Nanoprobes, Inc. Yaphank, New York Anthony Rhodes Senior Lecturer in Cellular Pathology Faculty of Applied Sciences University of the West of England Frenchay Campus Bristol, U.K. Kevin A. Roth Professor of Pathology, Director of Neuropathology The University of Alabama at Birmingham Birmingham, Alabama Veit Schubert Institute of Plant Genetics and Crop Plant Research Gatersleben, Germany Günter Schwamberger Research Scientist Department of Molecular Biology Division of Allergy and Immunology University of Salzburg Salzburg, Austria Dietmar Schwertner digitaloptics Jena, Germany
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Michael Schwertner Department of Engineering Science University of Oxford Oxford, U.K.
Chris M. van der Loos Department of Cardiovascular Pathology Academic Medical Center Amsterdam, the Netherlands
Marek Skacel Associate Staff Department of Anatomic Pathology The Cleveland Clinic Foundation Cleveland, Ohio
Leo Wollweber Institute of Molecular Biotechnology Department Single-Cell and SingleMolecule Techniques Jena/Thueringen, Germany
Sabine Tontsch IVF Center, Department of Gynecology and Obstetrics St. Johanns-Hospital, Salzburger Landeskliniken Salzburg, Austria
Masahiko Zuka Research Associate Department of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, California
Raymond R. Tubbs Section Head for Molecular Genetic Pathology Professor and Chairman Department of Clinical Pathology and the Cleveland Clinic Lerner College of Medicine The Cleveland Clinic Foundation Cleveland, Ohio
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CONTENTS 1
Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens in Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Guida M. Portela-Gomes
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Multiple Staining in Molecular Morphology . . . . . . . . . . . . . . . . . . . . . . . .27 Chris M. van der Loos
3
Enzyme-Based Fluorescence Amplification for Immunohistochemistry and In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65 Kevin A. Roth and Denis G. Baskin
4
Gold Cluster Labels and Related Technologies in Molecular Morphology. . 81 James F. Hainfeld and Richard D. Powell
5
Gold- and Silver-Facilitated Metallographic In Situ Hybridization Procedures for Detection of HER2 Gene Amplification . . . . . . . . . . . . . .101 Raymond R. Tubbs, James Pettay, Marek Skacel, Erinn Downs-Kelly, Richard D. Powell, David G. Hicks, and James F. Hainfeld
6
Toward Molecular Sensitivity: Tyramide Signal Amplification in Molecular Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107 Gerhard W. Hacker, James Pettay, and Raymond R. Tubbs
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Array-Based Comparative Genomic Hybridization as a Tool for Survey of Genomic Alterations in Human Neoplasms . . . . . . . . . . . . . . . . . . . . .123 Dina Kandil, Marek Skacel, James D. Pettay, and Raymond R. Tubbs
8
Southwestern Histochemistry: A Method for Localization of Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .133 Shin-ichi Izumi, Takehiko Koji, and Paul K. Nakane
9
High Throughput Morphological Gene Expression Studies Using Automated mRNA In Situ Hybridization Applications and Tissue Microarrays for Post-Genomic and Clinical Research . . . . . . . . . . . . . . . .147 Hiro Nitta, David G. Hicks, Marek Skacel, James D. Pettay, Thomas Grogan, and Raymond R. Tubbs
10 Whole-Mount In Situ Hybridization: Manual and Automated Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157 Sabine Tontsch, Günter Lepperdinger, Isabella Artner, and Hans-Christian Bauer 11 An Open Door to Molecular Cytopathology: Interphase Fluorescence In Situ Hybridization (FISH) on Liquid-Based Thin-Layer Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .167 Marek Skacel, James D. Pettay, Marybeth Hartke, and Raymond R. Tubbs 12 Recent Developments in In Situ PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . .179 Lisa Bobroski and Omar Bagasra xxi
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13 High-Resolution Visualization of Apoptotic Markers in Human Biopsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199 Andreas P. Aschoff and Gustav F. Jirikowski 14 Three-Dimensional Full Color Demonstration of Bright-Field and Fluorescence Microscopic Preparations: The Digital Optical Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209 Gerhard W. Hacker, Veit Schubert, Leo Wollweber, Michael Schwertner, and Dietmar Schwertner 15 Visualization of Signal Transduction Pathways in Real Time: Protein Kinase C and Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .225 Michelle R. Lennartz, Pamela M. Brannock, and Joseph E. Mazurkiewicz 16 Proteolytic Activity Demonstrated by Film In Situ Zymography (FIZ): A Clinically Applicable Double-Staining Method Also Involving ImmunoGold-Silver Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245 Masahiko Zuka 17 Conscious Production and Purchase of Reagents for Molecular Morphology: Methodological, Ethical, and Legal Considerations . . . . . . .253 Gerhard W. Hacker, Antoine F. Goetschel, and Günter Schwamberger 18 Quality Assurance of Immunocytochemistry and Molecular Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .275 Anthony Rhodes Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .295
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens in Light Microscopy Guida M. Portela-Gomes
1.1 INTRODUCTION Immunostaining techniques are widely used for co-localization studies of multiple peptide antigens. In this chapter different immunohistochemical staining methods are reviewed and their advantages and disadvantages are discussed. Single immunostaining on consecutive sections is briefly evaluated. Particular attention is paid to methods for double or triple immunostaining on the same section, both for brightfield light microscopy (elution and nonelution techniques) and fluorescence microscopy (including methods to avoid cross-reactivity of primary and secondary antibodies). Various fluorophore properties that may influence their choice in a given staining procedure are commented. In addition, methods for quadruple immunostaining on the same section are mentioned. For co-localization studies, multiple immunostaining using fluorophores usually have more advantages than the other methods, and protocols are suggested in Section 1.5. The development of biochemical, molecular genetical, and histochemical tech0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
niques has led to the identification and characterization of a great number of biological substances. An increasing number of reports show that one single cell type may synthesize several proteins. A knowledge of the physiological functions of many of these proteins is still limited, especially regarding their functional interactions. Therefore, a simultaneous intracellular visualization and localization of the substances on tissue sections is an important first step in investigating their functional interrelationships. The aim of this chapter is to give an overview of the different methods available for immunohistochemical co-localization of different proteins in bright-field and fluorescence microscopy.
1.2 SINGLE IMMUNOSTAINING ON CONSECUTIVE SECTIONS Single immunostaining performed on consecutive sections is an accurate method for light microscopical co-localization studies of two antigens in relatively large structures (for example, cells, either in cryostat
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Molecular Morphology in Human Tissues: Techniques and Applications [1] or in paraffin sections [2, 3]). However, this method is not widely used for thin structures, such as nerve fibers, as they are too thin compared to the thickness of the sections, and the fibers would not be present in both consecutive sections. Even for studies of single cells, it can be difficult to identify their cellular parts in the respective section, even if mirror image sections are used. This difficulty decreases by using thin sections, 1 to 2 µm thick, but on the other hand, there are less structures that are stained and thus the staining intensity decreases. It is therefore possible that antigens that are present in very small amounts may not be demonstrated. With thicker sections there are limits in the penetration of the antibodies in the tissue sections, which may give differences in consecutive non-mirror image sections. When using mirror image sections, the evaluation of the staining results is facilitated by printing in reverse the negative film of one of the sections so that the sections will be seen as a serial section (Figure 1.1{1}). If very thin sections are used, it is possible to perform single immunostaining in three consecutive sections, but it is difficult in practice to demonstrate more than two substances in the same cell using a single immunostaining technique.
1.3 DOUBLE OR TRIPLE IMMUNOSTAINING ON THE SAME SECTION 1.3.1
Bright-Field Light Microscopy
1.3.1.1 Elution Techniques The use of immunostainings for the identification of three tissue antigens in the same section was introduced by Nakane [4], who visualized simultaneously three
2
hormones in the anterior pituitary gland using sequential stainings, demonstrated with three different peroxidase chromogens. With elution techniques, immunoenzymatic methods are usually used in sequential staining. After the initial staining, the entire antigen–antibody complex is eluted (i.e., removed from the tissue section), either with an acid buffer, such as glycinehydrochloric acid [4], with unbuffered hydrochloric acid [4, 5], or with a strong oxidizing reaction (e.g., potassium permanganate with sulfuric acid [6]), but the enzyme reaction product remains. Thereafter, the second immunostaining is performed with either the same enzyme or a different one, but using another chromogen, giving a reaction product of a different color. The sections are usually photographed after the initial staining sequence (and the second sequence in triple staining) for comparison of the same structures after the second (and third) staining sequence. 1.3.1.1.1 Immunoenzymatic Methods with the Same Enzyme Horseradish peroxidase is the enzyme most frequently used in immunohistochemical labeling. Different chromogens can be used to visualize peroxidase, resulting in a precipitated dye [7]. The demonstration of peroxidase with the 3,3′-diaminobenzidine tetrahydrochloride (DAB)–hydrogen peroxide reaction [8] is probably the most commonly used. Briefly, horseradish peroxidase in the presence of its substrate, hydrogen peroxide, catalyzes the oxidation of an electron donor, DAB (co-substrate). On oxidation, the free bonds of DAB form an insoluble brown phenazine polymer that is deposited on the peroxidase enzymatic reaction site. This polymer can chelate with heavy metals. Thus, the brown color of the
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens
FIGURE 1.1 (Color Figure 1.1 follows page 106.) (1) Consecutive rat antral sections stained with the PAP technique using polyclonal serotonin antiserum (A) and polyclonal antiserum neutralized with tetragastrin (B). The arrows indicate some cells that were stained when neutralized antiserum was used. (Bar = 65 µm.) (2) Rat antral mucosa double stained for gastrin with the immunogold-silver staining method (brown-black) and for serotonin with the avidin–biotin complex method with amino-ethylcarbazole as chromogen (red). (Bar = 65 µm.) (3) Diagram representing the three ground colors emitted by the usual fluorophores, as observed with the commercially available fluorescence microscope filters (green, red, and blue), and the additive colors resulting from their co-localization. The co-localization of green and red is yellow, green and blue is cyan, red and blue is magenta; and the co-localization of all three ground colors is white. (4) Human pancreatic islet double-immunostained for synaptic vesicle protein 2 (SV2; Texas Red) and insulin (FITC). The microphotograph, taken using a double-band filter set, demonstrates that SV2 is present in all insulin cells, which is illustrated by the yellow color. Non-insulin cells are also immunostained (red). (Bar = 27 µm.) (5) Human antral mucosa triple stained with indirect immunofluorescence techniques for: (A) gastrin (AMCA), (B) chromogranin A (FITC), and (C) chromogranin B (Texas Red). (D) Double exposure through the double-band filter for FITC and Texas Red and through the AMCA filter is shown. Most gastrin cells (blue) (A) show stronger immunoreactivity for chromogranin B (red) (C) than for chromogranin A (green) (B), evident as varying intensities of magenta color (D). A few gastrin cells are more strongly immunoreactive to chromogranin A, visualized as cyan color (D). The white cells in D reflect the co-localization of the three fluorochromes together. (Bar = 27 µm.) (From Portela-Gomes, G.M., Stridsberg, M., Johansson, H., and Grimelius, L., J. Histochem. Cytochem., 45(6), 815–822, 1997. With permission.)
3
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Molecular Morphology in Human Tissues: Techniques and Applications peroxidase reaction product of DAB can turn to black by additional treatment of the sections with heavy metals, such as cobalt chloride [9], osmium tetroxide [8, 10], copper sulfate [11], gold chloride [12], or nickel ammonium sulfate [13, 14]. A black DAB reaction product can also be obtained using nickel ammonium sulfate together with DAB, having glucose oxidase as substrate; in this reaction, hydrogen peroxide, necessary for the oxidation of DAB, is formed by the action of glucose oxidase on glucose [15]. This black color gives a good contrast to the brown DAB color in double immunostaining. Other chromogens (electron donors) can also be used for the visualization of peroxidase; for example, 3-amino9-ethylcarbazole (red color) [16] or 4-chloro-1-naphthol (grayish-blue color) [4], which are soluble in ethanol, and αnaphthol–pyronin (reddish-pink) [17], odianisine (green) [18], or p-phenylenediamine and pyrocatechol (Hanker-Yates reagent, blue or brown-black color) [19, 20], which are insoluble. At present, there are several commercially available peroxidase substrate solutions, giving reaction products with these colors and others, such as Vector® VIP chromogen, giving a violet color (Vector Laboratories, Burlingame, CA). However, the sensitivity of the detection with DAB is greater than that obtained with any of these other chromogens [21]. 1.3.1.1.2 Immunoenzymatic Methods with Different Enzymes When two different enzymes are used in the staining process, usually horseradish peroxidase is used in combination with calf intestine alkaline phosphatase [22], but combination with Aspergillus niger glucose oxidase [23] or Escherichia coli β-D-galactosidase [24] can also be used. Alkaline phosphatase activity can be visualized with the substrate naphthol AS-MX (3-hydroxy2-naphthoic acid 2,4-dimethylanilide) 4
phosphate in conjunction with a diazonium salt, Fast Red TR, or Fast Blue BBN; alkaline phosphatase hydrolyzes the naphthol phosphate ester and produces a phenolic compound that couples to the diazonium salt, giving, respectively, a red or blue-purple ethanol-soluble azo dye [22, 25]. Alkaline phosphatase can also be demonstrated with the substrate naphthol AS-BI phosphate using New Fuchsin as the coupling salt, which gives an insoluble red reaction product with a stronger color than Fast Red TR [26]. Alkaline phosphatase substrate kits are available, giving red, blue, or black insoluble reaction products. Glucose oxidase has a flavin as the prosthetic group, which catalyzes the oxidation of glucose forming hydrogen peroxide. Then, glucose oxidase transfers hydrogen to a colorless tetrazolium salt, producing the reduced reaction product formazan, which is intensely colored; nitroblue tetrazolium (NBT; navy-blue color) and 2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT; brick-red color) formazans are ethanol soluble, while the reaction product of tetranitroblue tetrazolium (TNBT; brown) is insoluble [27, 28]. Glucose oxidase kits are also available (Vector Laboratories). β-D-Galactosidase catalyzes the hydrolysis of the substrate 5bromo-4 chloro-3-indolyl-β-D-galactoside and produces galactose, giving, on oxidation of ferro-ferricyanide, a turquoise-blue indigo polymer [29, 30], and on oxidation of nitroblue tetrazolium, a blue-purple formazan [31], both reaction products insoluble. This latter enzyme can also be detected with commercially available substrates yielding magenta or blue-gray products (Molecular Probes, Eugene, OR). When using enzymatic methods, inhibition of the respective endogenous enzyme activity should be performed to avoid falsepositive reactions. Endogenous peroxidase is found in leucocytes and erythrocytes,
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens and its activity is most commonly abolished by preincubation of the sections by water, buffer, or absolute methanol containing hydrogen peroxide. Endogenous alkaline phosphatase is usually not a problem in routinely fixed, paraffin-embedded specimens. If necessary, adding levamisole to the substrate solutions blocks endogenous alkaline phosphatase, except in the intestine. Intestinal alkaline phosphatase can be blocked by preincubation of the sections with 20% acetic acid, or with a mixture of 0.3% hydrogen peroxide and 2.5% periodic acid, but these acidic pretreatments may damage the tissue antigens. Glucose oxidase is not present in mammalian tissues and Escherichia coli β-Dgalactosidase is a bacterial enzyme that reacts at a different pH than the mammalian enzyme. In multiple staining it is important to choose the sequence in which the chromogens and their substrates are used. Nakane [4] determined that DAB, αnaphthol-pyronin and 4-chloro-1-naphthol was the optimal sequence in his stainings. DAB is usually the first choice to recommend, leaving the choice of an ethanol-soluble substrate for the last staining sequence. When using elution techniques, careful controls must be performed to check for complete detachment of the primary and secondary antibodies from the sections, in order to exclude cross-reactivity between the staining sequences and thus ensure staining specificity. The time for the elution solutions to dissociate completely the antigen–antibody complexes varies with different antigens and must be tested for the individual antibodies. However, one disadvantage of the elution techniques is that there are difficulties in removing antibodies of high avidity,
which are strongly attached to antigens. In these cases, the first primary antibody continues to react with the secondary antibodies of the second staining sequence. Therefore, elution techniques may be useful when using antibodies of low avidity. The secondary peroxidase-labeled antibodies of the first staining sequence may also react with the substrate of the second staining sequence. Another disadvantage of elution techniques is that the antigen epitopes may be destroyed or changed during the elution. The most efficient and reliable elution solution seems to be a mixture of potassium permanganate and sulfuric acid [6]; however, it has been proposed that this oxidation procedure destroys antigenic binding sites containing methionine, tryptophan, and disulfide groups [32]. In double staining for bright-field microscopy, care must be exercised in the choice of chromogens so that the differences in the light spectrum are enough to give a good color contrast. Double or triple stainings with chromogens for light microscopy are excellent if the substances in question occur in different cells; but if they are localized in the same cells, it is difficult to evaluate the mixed-color product. This difficulty increases if one antigen occurs in greater quantity than the other(s). In this case, a strong reaction with one chromogen may hide a weak reaction with another chromogen. This problem is minimized by carrying out elution of both the antibodies and the reaction product of the first staining sequence, before performing the second staining procedure. In this case, an ethanolsoluble reaction product must be used (e.g., chloro-naphthol, ethylcarbazole, Fast Blue, or Fast Red). These reaction products can be removed, after the elution of the antibodies, by immersion of the sections in absolute ethanol; however, part of the reaction product may have already been removed at the same time as the antibodies 5
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Molecular Morphology in Human Tissues: Techniques and Applications during the elution procedure. Thus, co-localization can be demonstrated in the same section but not at the same time. This procedure requires careful controls, and microphotographs at least after the initial staining. It is also possible that the first reaction product blocks or interferes with the second, and it may be that there is residual peroxidase from the first staining sequence that might continue to react with the second developing solution. 1.3.1.2 Non-Elution Techniques 1.3.1.2.1 Labeled Primary Antibodies Direct conjugation of primary antibodies with bright-field light microscopy labels of different types, such as enzymes, gold particles (colloidal gold or Nanogold™) (Nanoprobes, Inc., Yaphank, NY; Aurion, Wageningen, the Netherlands), or radioactive substances allows the use of antibodies raised in the same animal species without antibody cross-reaction problems. This direct method has a high specificity, as the primary antibodies will not interfere with one another; however, it has lower sensitivity compared with indirect techniques as it is possible to bind only one of the marker molecules to each primary antibody molecule [33]. The more recent Enhanced Polymer One-Step (EPOS; DakoCytomation, Glostrup, Denmark) system [34], which uses a dextran polymer “backbone” labeled with several peroxidase molecules, makes it possible to attach a large number of peroxidase molecules directly to the primary antibody. This EPOS visualization system has been successfully used in double staining in combination with immunostaining with alkaline phosphatase or β-galactosidase [35, 36]. However, the molecular size of the anti6
body is important for its penetration into the tissue; therefore, dextran’s high molecular weight may limit the penetration in the tissue section. Furthermore, by using labeled primary antibodies, a larger amount of the antibodies is usually needed to get optimal staining results and the preparation technique is more time consuming. However, recently, the direct labeling of small amounts of a primary mouse antibody with biotin or an enzyme (peroxidase or alkaline phosphatase) can be performed using commercially available kits (ZenonTM reagents; Molecular Probes); this is an efficient, easy, and rapid procedure. In the direct methods, there is also a risk of changing the antibody properties. 1.3.1.2.2 Double–PAP Staining Double immunoperoxidase staining, using the peroxidase–anti-peroxidase (PAP) technique [37] with DAB as the first chromogen, was performed for the first time by Sternberger and Joseph [38, 39], using a very strong peroxidase reaction, which was achieved with both a high DAB concentration and a long reaction time. By using a strong reaction, a high concentration of polymeric oxidized DAB deposits was formed. These DAB deposits masked the antigenic binding sites of the antibodies of the first staining sequence, and thus blocked any further reaction with the antibodies of the second staining sequence. In the second staining sequence, a chromogen giving a color contrast was used. In this procedure, DAB with heavy metal intensification in the first staining sequence, followed by DAB alone in the second sequence, can be used [12]; but other peroxidase chromogens can also be used in the second staining sequence, with reaction products giving a color contrast to either the brown or the black DAB color (see Sec-
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens tion 1.3.1.1.1). A triple–PAP staining can also be performed using DAB with heavy metal intensification in the first staining sequence, followed by DAB alone in the second sequence, and 4-chloro-1-naphthol in the third [12]. This double–PAP technique is only useful if the two antigens in question are localized in different cells. When the antigens are present in the same cell, significant problems can appear in evaluating the color product. Furthermore, when the DAB reaction product is strong, immunologic or catalytic interactions can occur with the second sequence antibodies or substrates [40]. 1.3.1.2.3 Double Immunostaining with Peroxidase in Combination with Other Labels Peroxidase has been used in different methods as an important link in immunostaining, originally in the PAP technique [37] and later in the more sensitive avidin–biotin complex (ABC) method [41]. In the ABC method, a biotinylated secondary antibody is applied, followed by incubation with ABC. Biotinylation does not affect the affinity of the immunoglobulin (Ig) G; and biotin, being a small molecule, does not change the size of the IgG molecule, which is important for its penetration into the tissue. The sensitivity of the ABC method is further due to enhancement by avidin; the avidin molecule has four high-affinity binding sites for biotin, which bind noncovalently to each other [42], thus amplifying the detection and increasing sensitivity. However, in addition to the general unspecific background problems, the ABC method may give rise to some specific background staining. This can be partly related to the basic isoelectric point of avidin (10.5) that caus-
es an unspecific binding to negatively charged tissue structures (e.g., nuclei or cell membranes). This problem is prevented using streptavidin instead of avidin, as streptavidin has a neutral isoelectric point. Another reason for the background staining with the ABC technique is related to endogenous biotin, which is present in many tissues and will react with avidin and streptavidin. This unwanted staining can be minimized by blocking endogenous biotin by pretreatment of the sections with avidin for 15 min, followed by biotin for 15 min for saturation of the free biotin binding sites of avidin (Vector Laboratories), but the results with this procedure are not always consistent (own observations). The sensitivity of the (strep)avidin–biotin complex (S-ABC) technique can be further increased using additional treatment with heavy metals (see Section 1.3.1.1.1), or by using amplification with the catalyzed reporter deposition (CARD) or catalyzed signal amplification (CSA) method (see Section 1.3.2.2.1.3). With the CARD method, care must be exercised to avoid background staining. Less background is present when using an indirect non-biotin amplification method (CSA II, DakoCytomation), which employs a fluoresceinlabeled tyramide and an anti-fluorescein peroxidase-labeled IgG that can be visualized by DAB. However, this system is, so far, only available for mouse monoclonal primary antibodies. Peroxidase is also used in the indirect two-step dextran polymer technique (EnVision™ system, DakoCytomation) [43], which uses a secondary antibody conjugated with a dextran polymer “backbone” labeled with a large number of peroxidase molecules. This technique has usually a high sensitivity of detection, it gives less background staining than the SABC technique because it has neither biotin nor avidin, but it has been reported 7
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Molecular Morphology in Human Tissues: Techniques and Applications to give a varying sensitivity in certain cases due to dextran’s molecular weight, which limits its penetration into the tissue [44]. More recently, a large number of peroxidase molecules have been attached to a secondary antibody by means of polymerizable molecules with a small molecular size (PowerVision; ImmunoVision Technologies Co., Daly City, CA) [45], thus giving an increased sensitivity to the polymer technique.
another antigen occurring at a high concentration is simultaneously visualized. Careful controls are necessary to ensure staining specificity, including the reversal of the staining sequences. A total separation of the colors of the mixed reaction has been reported with success, using digitized images with an image analysis system [46]; however, the possibility of interference of the first reaction product with the second cannot be excluded.
The S-ABC and the dextran polymer techniques can be performed either manually, using staining kits from the manufacturers, or by means of automated immunohistochemistry staining systems available from Ventana Medical Systems, Tucson, AR (e.g., NexES or BenchMarck XT) and DakoCytomation, Carpinteria, CA (Autostainer), respectively. Ventana NexES instruments employ staining under pressure and controlled heating, conditions that give an important decrease of background but that cannot be applied to all cases; however, more recent instruments (BenchMark XT) are flexible, having varying temperature settings.
Alkaline phosphatase, the enzyme most usually employed in combination with peroxidase, is available either conjugated to an IgG or to streptavidin, and in combination with anti-alkaline phosphatase (APAAP complex) [25], or with labeled polymer. These two latter methods are as sensitive as the S-ABC technique. Glucose oxidase conjugated to an IgG has a lower sensitivity, but this can be increased by using glucose oxidase–anti-glucose oxidase (GAG) complexes [47, 48]. A triple immunoenzymatic staining has been reported using a combination of peroxidase (red color), alkaline phosphatase (blue), and β-galactosidase (green) [49].
Peroxidase is also used in the non-biotin amplification technique (NBATM; Zymed Laboratories Inc., San Francisco, CA), in which a fluorescein isothiocyanate (FITC) secondary antibody is applied, followed by incubation with an anti-FITC horseradish peroxidase–conjugated tertiary antibody.
Non-enzymatic labels such as gold and silver particles, radioactive substances, or fluorophores (see Section 1.3.2.3) can be successfully used in double staining.
Different combinations of double staining have been reported, often using one immunoperoxidase method with DAB in the first sequence, and in the second sequence either other enzyme and chromogen or non-enzymatic markers. These techniques also have all the problems of evaluation of the color of the mixed reaction product, and they are not suitable for detecting small amounts of antigen if 8
1.3.1.2.3.1 Combination with Gold Particles Colloidal gold was initially used in double staining as a label for secondary antibodies, employing gold particles of a diameter of 15 to 20 nm, which are visible in bright-field light microscopy giving a reddish or deep pink color [50]. This method had a low sensitivity as these larger gold particles have poor penetration in the tissue. A higher sensitivity was achieved with colloidal gold particles of smaller diameters (1 to 5 nm), which are,
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens however, not visible with the light microscope. But using a silver or gold enhancement process (autometallography), the size of the gold particles increases to a visible size, which appears as a distinct black product [51, 52]. A further improvement of the sensitivity of this immunogold-silver staining (IGSS) method was achieved with the introduction of Nanogold-conjugated antibodies or streptavidin [53]. Double immunostaining can be performed using the IGSS method in the first staining sequence, followed by staining with enzymes, giving reaction products of contrasting colors, such as β-galactosidase (turquoise-blue reaction product) [54], horseradish peroxidase visualized with ethyl carbazole (red [55]; Figure 1.1{2}), or alkaline phosphatase (red or blue [56]). Alkaline phosphatase, detected with Fast Red in conjunction with naphthol AS-MX gives a reaction product that appears red with bright-field microscopy but also gives a strong red fluorescence in fluorescence microscopy [57]. By combining IGSS with this staining, there are no problems in evaluating the colors of the two chromogens, as they can be visualized separately with bright-field and fluorescence microscopy [58]. For further information, see Section 1.3.2.5.1. However, strong gold–silver precipitates may mask the antigenic binding sites of the antibodies of the first staining sequence in a similar way as described above for the double–PAP staining. It is also possible that interactions between silver precipitates and the second staining sequence may give rise to unspecific staining, as mentioned for DAB. 1.3.1.2.3.2 Combination with Silver Particles Colloidal silver has also been applied in double staining. It gives a light yellow color, visible in bright-field microscopy
[59] with a very weak contrast, and therefore it is very rarely used. 1.3.1.2.3.3 Combination with Radioactive Labels The simultaneous visualization of two antigens has been carried out using an immunoperoxidase staining for the first antigen, followed by radiolabeled (tritiated or iodinated) primary or secondary antibodies to identify the second antigen by means of autoradiography [60]. Radioimmunohistochemistry [61] has been used, particularly in studies of the nervous system [62]. Autoradiography has also been used in double and in triple staining, together with fluorescent labels [63, 64]. However, although radioactive labels are easy to quantify, they are not commonly used in immunohistochemistry because they have disadvantages. The radiolabeled antibodies must be carefully handled to avoid contamination, and the short halflife of some of these isotopes limits their usefulness. Furthermore, the development of the autoradiographs takes several days and the method gives relatively weak morphological resolution. There are also a limited number of commercially available radiolabeled antibodies compared to antibodies labeled with the other labels mentioned above.
1.3.2
Fluorescence Microscopy
1.3.2.1 General Immunofluorescence Methods Double immunofluorescence methods were described in 1957 by Silverstein, using antibodies labeled with fluorophores of two different colors: fluorescein isothiocyanate (FITC) and rhodamine B [65]. Different immunofluorescence staining methods have been described. The direct 9
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Molecular Morphology in Human Tissues: Techniques and Applications immunofluorescence technique was introduced by Coons and Kaplan in 1950 [66]. This method, using labeling of primary antibodies, has disadvantages already mentioned (see Section 1.3.1.2.1). However, for mouse monoclonal antibodies, the recently introduced Zenon labeling kits (Molecular Probes) provide a fast and reliable procedure for labeling very small amounts of antibody. With this technique, different fluorophore-labeled goat Fab fragments raised against the Fc portion of mouse IgG of one of three isotypes (IgG1, IgG2a, or IgG2b) can be used; it thus allows triple staining with three mouse primary antibodies provided they are of different isotypes. Although with this technology the fluorescence intensity can be adjusted by varying the extent of antibody labeling, the intensity is usually lower than that obtained with the indirect technique (own observations). The indirect technique [67], where the secondary antibodies are labeled with a fluorophore, is more sensitive than the direct one. In the indirect technique, the dilution of the primary antibody is higher than that in the direct technique, but lower than with immunoperoxidase techniques. However, by using the fluorophore-labeled streptavidin–biotin complex (S-ABC) method (see Section 1.6, Protocol I), higher dilutions of the primary antibody can be used. This fluorophorelabeled S-ABC method combines the sensitivity of the enzymatic and fluorescence procedures. Thus, while enzymatic methods have a high sensitivity because many substrate molecules are detected as a visible reaction product at the antigenic site, fluorescence has a higher detection sensitivity than absorbance in light microscopy. The sensitivity of the S-ABC method can be further increased by the catalyzed reporter deposition (CARD) method, either directly using a fluorophore-conjugated 10
tyramide, or indirectly using a biotinlabeled tyramide visualized by fluorophore-conjugated streptavidin [68, 69]. The CARD method has also been termed “tyramide signal amplification” (TSA; Perkin-Elmer Life Sciences Products, Boston, MA) or the “catalyzed signal amplification” (CSA; DakoCytomation) method. Amplification with the CARD method is particularly useful in co-localization studies when the antigen is present in a small quantity; this staining technique is useful in double-immunofluorescence stainings [70–73] (see Section 1.6, Protocols IV and V). In co-localization studies, immunofluorescence techniques, used in double or triple staining performed on the same section, are superior to all other techniques because the substances can be determined more exactly and without any interference in individual cells. The evaluation of the staining can also be performed at the same time. There is also no mixing of colors if proper fluorescence microscope filters are used. 1.3.2.2 Methods to Avoid CrossReactivity of the Antibodies 1.3.2.2.1 Cross-Reactivity of Primary Antibodies With double or triple immunostaining, cross-reactivity of the antibodies must be prevented. Therefore, primary antibodies raised in the same animal species should be avoided. One primary monoclonal antibody can be successfully mixed with two polyclonal antibodies if the latter antibodies are raised in different animal species (e.g., rabbit and either guinea pig, chicken, sheep, or goat). Two monoclonal antibodies, raised in rat and mouse, respectively, can also be used together with a polyclonal antibody. Thus, a “one-staining sequence”
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens procedure can be carried out by incubating the sections with a cocktail of primary antibodies followed by an incubation of a mixture of the species-specific fluorophorelabeled secondary antibodies. This is the most simple and reliable multiple staining procedure [74–78]. However, two primary antibodies raised in the same animal species can be used. As mentioned in Section 1.3.2.1, two directly labeled mouse primary antibodies of different isotypes can also be reliably mixed with one polyclonal antibody. With other primary antibodies raised in the same animal species, care must be exercised to prevent the primary antibody of the second staining sequence from reacting with free binding sites of the secondary antibodies of the first staining sequence, but also to prevent the primary antibodies from the first staining sequence from reacting with the secondary antibodies of the second staining sequence. Several methods have been proposed to handle this situation; three reliable techniques are discussed now. 1.3.2.2.1.1 Use of Monovalent Fab Fragments of IgG The epitopes of the primary antibodies are saturated using fluorochrome-labeled monovalent Fab fragments of IgG as the second layer of antibodies [74, 79] (see Section 1.5, Protocol III). Fab fragments of IgG, obtained by papain proteolysis, are univalent for antigen binding; that is, they have a single antigen binding site, contrary to F(ab)2 fragments, obtained by pepsin digestion, which are divalent as the whole IgG molecule. Thus, the monovalent characteristic of these Fab secondary antibodies, applied in the first staining sequence, blocks the possibility of any further reaction with the primary antibodies of the second staining sequence. Using this method, the antigenicity of the tissue is not destroyed or masked. This is the most effi-
cient method, provided that the concentration of the Fab antibodies and the incubation time are carefully tested to ensure saturation, and therefore give a blocking effect to the epitopes of the primary antibody. In our studies, an Fab concentration of 0.2 mg/ml overnight was found to saturate the epitopes of the first-step antibodies [74]. 1.3.2.2.1.2 Use of Paraformaldehyde Vapor When double immunostaining with primary antibodies raised in the same animal species, the epitopes of the conjugated secondary antibodies of the first staining sequence can be selectively destroyed by exposure to paraformaldehyde vapor at 80°C [80]. In this procedure it is important to obtain a complete saturation of the anti-IgG binding sites of the first primary antibody and therefore it is necessary to use optimally diluted primary antibodies in order to get saturation of its epitopes by the secondary antibodies. Then, denaturation of the free antigen binding sites of the IgG is obtained by paraformaldehyde vapor. The time for a complete denaturation varies with section thickness; in 5µm-thick paraffin sections, usually 4 hr of paraformaldehyde treatment are necessary [70, 75]. This method is simple but it is not suitable for antigens that are denaturated by formaldehyde, such as glucagon and synaptophysin (personal observations). 1.3.2.2.1.3 Use of CARD-Based Methods
•
CARD method followed by indirect immunofluorescence: For double staining with primary antibodies raised in the same species, the horseradish peroxidase catalyzed deposition of tyramide (CARD method) can be used initially in the staining sequence. The first approach of this procedure was reported using a high dilution of the first primary antibody, which was 11
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Molecular Morphology in Human Tissues: Techniques and Applications
•
•
12
visualized by the fluorescent CARD method. In the second staining sequence, an indirect immunofluorescence technique was used with another fluorophore [81, 82]. This staining procedure was based on the principle that the highly diluted antibodies of the first step cannot be demonstrated by an indirect immunofluorescence technique, but it is possible to detect them with the CARD amplification. Therefore, the former technique can be safely used after CARD. Triple-CARD with elution: A tripleCARD procedure has also been reported in order to visualize three DNA probes, with three tyramide detection systems labeled with three different fluorophores, where an elution with hydrochloric acid was carried out between each staining procedure [83]. Double-CARD without elution: Horseradish peroxidase reacts with hydrogen peroxide and catalyzes the oxidation of the phenol group of tyramide, resulting in the formation of a quinone-like compound with a short half-life radical on the C2 group. The oxidized and radicalized tyramide binds covalently to tyrosine residues and other electronrich amino acids in the tissue at the site of the peroxidase enzymatic reaction [69, 84]. It has been shown that these tyramide deposits block any further cross-reaction with the second staining sequence, as well as that tyramide deposition in tissues does not influence the antigenicity of the second epitopes, as does DAB deposition [82]. Thus, a double-CARD method, using biotinylated tyramide in both staining sequences, has also been successfully employed for immunohistochemical co-localization studies, without carrying out any elution. In this case, due to the high affinity of the
four binding sites of the avidin molecule to biotin, the biotin of the first staining sequence must be saturated by incubation with unlabeled avidin. By this means, the binding of this first step biotin with the avidin of the second staining sequence is avoided. In our studies, an avidin concentration of 100 µg/ml overnight saturated the first-step biotin [73, 85]. 1.3.2.2.2 Cross-Reactivity with Secondary Antibodies In double or triple staining, cross-reactivity between secondary antibodies must also be avoided. Thus, care must be exercised in choosing secondary antibodies raised in different animal species. However, IgG of different animal species may show cross-reactivity with each other, especially in species similar to each other such as sheep and goat. But IgG from more different animal species, such as sheep or goat with guinea pig, may also cross-react [86]; another example would be sheep with rabbit IgG [87]. To ensure species specificity of the secondary antibodies, before application to the sections, the secondary antibodies of the first staining sequence should be preincubated, overnight at 4°C, with normal serum from the species donating the secondary antibodies of the second sequence and vice versa. The secondary antibodies should also be tested in relationship to the specificity of the species in which the primary antibodies have been raised; for example, a primary antibody raised in rabbit should be tested with a labeled secondary antibody against guinea pig. A further crossreaction problem may occur between the secondary antibody and IgG localized in the tissue, which can be avoided by preincubation of the secondary antibody with normal serum of the species recognized by the tissue; for example, in studies of rat tissue, the secondary antibodies should be preincubated with normal rat serum.
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens 1.3.2.3
Fluorophores
Fluorophores are usually polyaromatic hydrocarbons or heterocyclic molecules that are able to absorb photons (quantum of light), from a mercury lamp or laser, and emit them as photons of a longer wavelength (fluorescence) than the absorbed photons. This absorption/emission process can be always repeated if the fluorophores are not destroyed. A molecule that has absorbed a photon becomes an excited state molecule. In this excited state, there are electrons around the atomic nuclei of the molecule that are transferred from their ground (low-energy) orbital to a higherenergy orbital. These electrons return rapidly (∼10-8 seconds) to their normal lower-energy orbital, emitting fluorescence. The fluorescence photons have a lower energy, and thus a longer wavelength, due to energy dissipation. Each fluorophore absorbs only photons of a certain wavelength, as the absorbed photons must have the exact energy that corresponds to the energy used for the transfer of electrons to a higher-energy orbital. To emit the absorbed light as fluorescence, the fluorophores must have enough molecular rigidity to avoid dissipation of the energy. Thus, the excitation energy of an electronically excited molecule can be emitted as fluorescence, but it can also be dissipated in different forms, such as: • Transformation into heat (internal conversion to kinetic energy). • Fluorescence resonance energy transfer (FRET). FRET is a phenomenon in which there is transference of energy to an unexcited molecule at a near distance without emission of a photon. This nearby molecule, which must have an absorption wavelength within the emission wavelength of the donor, may emit fluorescence but at a wavelength that is both longer
and parallel to the emission wavelength of the donor molecule. This phenomenon has been used to increase the fluorescence from some fluorophores; for example, in flow cytometry, by mixing the fluorophores phycoerythrin (PE) and cyanine 5 (see below), there is a fourfold decrease of PE and an increase of cyanine 5 fluorescence due to energy transfer [88]. • Quenching (or fading). Fluorescence quenching is a photochemical reaction that results in a decrease in the fluorescence intensity without changing the emission wavelength. It can result from interactions between adjacent molecules leading to destruction of the excited fluorophore, or from the formation of non-fluorescent molecules. The chemical background for the quenching photochemical reaction is still primarily unknown, but some authors have proposed that oxygen, triplet states or protein denaturation might be involved [cf. 89]. All fluorophores fade under exposure to excitation light, depending on the intensity and the duration of illumination. Another, different phenomenon resulting from interaction between excited fluorophores may also occur, consisting of the formation of excimers, which are excited fluorophores that emit photons with alterations in the emission wavelength. Different fluorophores can be used for immunofluorescence. For co-localization studies, the choice of fluorophores is important, as they must provide a staining intensity with satisfactory brightness, as well as a distinct color for fluorescent detection. In addition, it is necessary to obtain a balance in the intensity of the fluorescent colors so that in simultaneous 13
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Molecular Morphology in Human Tissues: Techniques and Applications observations (see Section 1.3.2.4) one color does not dominate the others. This balance depends not only on the properties of the fluorophore per se, but also on the amount of antigen to be visualized. Thus, a stronger signal is necessary to visualize antigens present in small quantities. Various combinations of fluorophores can be used. The fluorophores that we prefer in double or triple immunofluorescence stainings when using mercury light sources are either FITC, Texas Red (TXRD), and 7-amino-4methylcoumarin-3-acetic acid (AMCA), or Alexa Fluor dyes 488, 594, and 350 (Molecular Probes), giving green, red, and blue fluorescence color, respectively. FITC (absorption/emission maxima (abs/em) 1 ∼ 494/520 nm) [66, 90] is one of the most widely used fluorophores. Although FITC has a rather rapid rate of fading ( i.e., decrease of fluorescence intensity) on exposure to ultraviolet light, it has in our studies given a bright green fluorescence with low background. Carbocyanine 2 (Cy 2; abs/em ∼ 492/510 nm) [91, 92], carboxyrhodol (Rhodol Green; abs/em 1 ∼ 499/525 nm) [93], and 2′-7′-difluorofluorescein derivatives (Oregon Green; abs/em ∼ 496–511/522–530 nm [94] have higher fluorescence intensities, and the two latter fluorophores are more stable to ultraviolet light than FITC [95]. The alkyl derivative of 4,4-difluoro-4-bora-3a,4adiaza-s-indacene (BODIPY FL; abs/em ∼ 503–512 nm) [96] can also be used as a green fluorophore, with greater photostability than FITC, but giving a less intense fluorescence. TXRD (sulphonyl chloride ester derivative of sulphorhodamine; abs/em ∼ 595/ 615–620 nm) [97, 98] gives a bright red fluorescence with very low background, and in double-staining shows virtually no overlapping with FITC. Other red fluorophores can also be used, such as tetram14
ethylrhodamine isothiocyanate (TRITC; abs/em ∼ 550–580 nm) [89] or indocarbocyanine 3.18 (Cy 3; abs/em ∼ 550–570 nm) [91, 100, 101]. TRITC has a lower fluorescence intensity than TXRD but is more stable to ultraviolet irradiation; however, it has an emission wavelength rather near FITC, which can give overlapping problems. Cy 3 gives a very strong red fluorescence [102] that, even at high dilutions, is therefore sometimes difficult to combine with other fluorophores, which must give a similar srength and brightness in order to allow a correct simultaneous visualization. Cy 3 has also been reported to show a yellow-green fluorescence, by using a specially constructed filter to select the appropriate wavelength [103]. Phycoerythrin (PE; abs/em ∼ 488-545/580) [104] is a fluorophore that gives an orangered fluorescence but it can be seen with an FITC filter as its emission wavelength is near to FITC. BODIPY TMR and TR (abs/em ∼ 542–589/574–617 nm) are red fluorophores with a great photostability. Indodicarbocyanine (Cy 5; abs/em ∼ 650/670 nm) [91, 100, 101] has a bright red fluorescence and is used for laser scanning confocal immunofluorescence microscopy. However, it is not used with the common mercury light sources because it has high absorption/emission wavelengths that are not suitable for observation with the human eye, which has a low sensitivity for high wavelengths within the red spectrum, but which can well be recorded with the confocal microscope. However, Cy 5 has recently been successfully used in multiple staining with a standard fluorescence microscope using a specially constructed filter to select the appropriate wavelength [103]. This filter is not yet commercially available. Regarding the blue fluorophores, AMCA (abs/em ∼ 345/445–450 nm) [105] displays a light blue color, which,
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens although fading rapidly, combines well with TXRD to produce a magenta color. Other blue fluorophores available include pyrenyloxytrisulphonic acid derivatives (Cascade Blue; abs/em ∼ 400/420–430 nm) [106], 6,8-difluoro-7-hydroxy-4methylcoumarin (Marina Blue, abs/em ∼ 365/460 nm), and 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue, abs/em ∼ 415/455 nm) [107], but we have not tested them. Recently, a new family of fluorophores was introduced by Molecular Probes, the Alexa dyes, which are available, among others, in IgG or avidin conjugates, giving fluorescence of different colors, namely, green, red, or blue as already mentioned. Alexa dyes are sulfonated rhodamine or coumarin derivatives; they give a stronger, brighter fluorescence and are more photostable than all other fluorophores available [108]. There are green and red Alexa fluorophores having a similar emission to FITC (Alexa Fluor 488; abs/em ∼ 495/519 nm) and TXRD (Alexa Fluor 594; abs/em ∼ 590/617 nm) or TRITC (Alexa Fluor 546; abs/em ∼ 556/574 nm), respectively. The Alexa Fluor 350 blue fluorophore (abs/em ∼ 346/442 nm), a sulphonated derivative of AMCA, has a shorter emission wavelength than AMCA, which decreases the overlapping with FITC. The new red fluorophore Alexa Fluor 610 (abs/em ∼ 612/628 nm) and the blue Alexa Fluor 405 (abs/em ∼ 402/421 nm) are claimed to give the best differentiation from the green fluorophores and to be the ideal choice for multiple staining. Based on this strong fluorescence, an Alexa-conjugated anti-fluorescein IgG molecule has also been introduced, a molecule suitable for amplification of the immunofluorescence staining that had been previously performed with fluorescein-labeled secondary antibodies. This amplification
procedure of the fluorophore is of special importance once FITC fades. There are fluorophores that change their fluorescence emission with different conditions. Some fluorophores are pH-sensitive (e.g., Oregon Green and seminaphthorhodafluors (SNARF; Molecular Probes)). This property makes them useful as sensitive pH indicators for studies of intracellular pH; for example, proteins tagged with these markers can be followed in living cells for detecting endocytosis into acidic organelles such as early endosomes. Oregon Green shows a marked decrease in fluorescence intensity in acidic pH (4.2–5.7). SNARF dyes have different excitation spectra at a more acidic or basic pH (range 6.0–8.0) with the correspondent pH-dependent emission wavelengths giving a yellowish or deep red fluorescence, respectively. Another fluorophore, DsRedE5, changes from an initial green emission fluorescence to yellow, orange, and then to red with time, over the course of about 16 hours. DsRed-E5 has been used as a fluorescent “timer,” or clock, to detect timedependent expression in evaluating promoter activity [109] or in following the movements of intracellular structures [110]; in these studies, recent structures appear green, older ones (with more than 16 hours) appear red, and those of intermediate age or with continuous activity appear yellow and orange, indicating the simultaneous presence of green and red fluorophores. More recently, water-soluble dyes (FM dyes; Molecular Probes) have been introduced as in vivo membrane markers for endo- and exocytosis studies. These FM dyes are non-fluorescent in aqueous media, but they turn fluorescent when attached to membranes and thereafter their movement inside cells can be followed. 15
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Molecular Morphology in Human Tissues: Techniques and Applications In immunofluorescence microscopy, with either mercury or laser light sources, the retardation of quenching, and a low background are important for accurate observations and microphotographs. • Quenching of Fading of the Excited Fluoroprobe The decay of fluorescence intensity on exposure to excitation irradiation is considerably retarded using mounting media containing anti-fading agents. Based on the principle that, when exposed to excitation light, a number of fluorophores are irreversibly oxidized and become nonfluorescent, the anti-fading mounting media contain substances that (1) retard the diffusion of oxygen, by increasing the viscosity of the medium (e.g., with glycerol or polyvinyl alcohol) and/or (2) retard or inhibit the generation of reactive oxygen species, thus decreasing the number of oxidized fluorophores; this can be achieved by using substances that quench the excitation or that act as free radical scavengers. There are various commercially available mounting media with substances that increase the fluorescence emission time, including 1,4-diazabicyclo[2.2.2]octane (DABCO; SlowFade Light, Molecular Probes); p-phenylenediamine (Vectashield, Vector Laboratories), an aliphatic tertiary diamine (fluorescence mounting medium, DakoCytomation); and other agents not specified by the suppliers (e.g., ProLong, Molecular Probes). The choice of mounting medium depends on its anti-fading capacity and on the intensity of fluorescence desired for each experimental condition. Mounting media with agents that quench excitation give a somewhat decreased initial fluorescence intensity [111]; however, this decrease can be minimized using a bright fluorophore (e.g., Alexa dyes), and there is little fading after a long exposure to illumination. These media have special advantages in confocal laser microscopy, when many scans can be neces16
sary. Anti-fading agents containing pphenylenediamine should be avoided when using cyanine dyes, as they can cleave cyanine, resulting in a decrease of fluorescence upon storage. The use of anti-fading agents can sometimes result in an increase in background fluorescence [112]. Storage of the slides in the dark and at +4°C or –20°C also helps to prevent fading. • Background Fluorescence Background fluorescence may be due to autofluorescence, that is, from the tissue components such as formaldehydeinduced fluorescence, but it depends mainly on non-specifically bound fluorophores. Background fluorescence occurs more often with ionic fluorophores because they are negatively charged and therefore will bind non-specifically to positively charged cell structures, such as proteins. FITC, cyanines, and Alexa dyes have a negative charge. To decrease background fluorescence, the nonspecific electrostatic binding of these fluorophores can be blocked, or decreased, by incubating the sections with 10% nonimmune serum from the animal species providing the fluorophore-conjugated antibodies, before applying the primary antibodies [70, 74, 75, 113]. In some cases, nonionic fluorophores, which have a neutral charge (such as BODIPY FL, TRITC, and TXRD) are preferable despite giving a lower fluorescence intensity. Background fluorescence can also be attributed, to some extent, to hydrophobic interactions; thus, adding 0.1 to 1% detergent (e.g., Tween-20 or Triton X-100) may help to reduce background when hydrophobic fluorophores are used. 1.3.2.4
Microscope Filters
Different standard fluorescence microscope filters are commercially available. A set of fluorescence microscope filters con-
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens sists of two filters and a dichroic mirror (beamsplitter). One filter, the primary or excitation filter, is placed between the light source and the tissue section. It is chosen so that it only transmits the light emitted from the light source with the selected wavelength that excites a given fluorophore. The dichroic mirror reflects this selected light and transmits it to the tissue section. The emitted fluorescence light is then reflected by the same mirror to the other filter, the secondary or barrier filter. This filter transmits only the light with the wavelength emitted when the fluorophore fluoresces, and does not allow the passage of any residual excitation light. It is important to use filters that select narrowband excitation and emission wavelengths of the different fluorophores, in order to ensure a specific identification of a given fluorophore, and avoid spectral overlap with the emission wavelength(s) of other fluorophore(s) in double or triple immunostaining. By using a double-band filter set (e.g., for FITC and TXRD), the co-localization of these two substances is seen as yellow (Figure 1.1{3 and 4}). Using triple staining techniques with FITC, TXRD and AMCA, or Alexa dyes 488, 594, and 350, as fluorophores, a triple filter is available, but this filter does not give distinct color combinations. Instead, we prefer to expose the photographic film with the double filter for green and red fluorescence, followed by double exposure using a single band filter for blue fluorescence. Thus, it is possible to identify a third substance at the same time. The co-localization of the blue fluorescence displayed by AMCA (or Alexa 350) with TXRD (or Alexa 594) gives a magenta color, AMCA (or Alexa 350) with FITC (or Alexa 488) cyan, while the co-localization of all three fluo-
rochromes together is seen as white (Figure 1.1{3 and 5}). 1.3.2.5 Fluorogenic Enzyme Substrates 1.3.2.5.1 Fluorogenic Alkaline Phosphatase Substrates Immunoenzymatic methods using alkaline phosphatase can be visualized with substrates that, upon enzymatic cleavage, form precipitates at the site of enzymatic activity, giving intense fluorescence; this procedure is called enzyme-labeled fluorescence (ELF) [114]. The fluorescence displayed by the ELF process is more photostable on ultraviolet irradiation than that emitted by the immunofluorescence fluorophores. The alkaline phosphatase substrate Fast Red TR with naphthol AS-MX gives a reaction product that emits a red fluorescence, and has been reported in double staining with the IGSS method as mentioned in Section 1.3.1.2.3.1 [57, 58]. The alkaline phosphatse substrate Fast Red K, developed by Ventana Medical Systems, displays both a bright red color in bright-field microscopy and a brilliant pink-red fluorescence, and has been used for protein detection in a triple staining procedure, together with double fluorescence in situ hybridization [115]. Another fluorogenic substrate for alkaline phosphatase is a quinazoline compound, 2′-(5′-chloro-2′phosphoryloxy-phenyl)-6-chloro-4-[3H] quinazolinone, known as ELF-97’ phosphatase substrate (Molecular Probes). ELF97’ gives a bright yellow-green fluorescence (abs/em ~ 345/530 nm) and its application has been reported in co-localization studies together with immunofluorescence, using red and blue fluorophores [116]. 1.3.2.5.2 Fluorogenic Peroxidase and βGalactosidase substrates Peroxidase, in the presence of hydrogen peroxide, catalyzes the oxidation of 10-acetyl-
17
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Molecular Morphology in Human Tissues: Techniques and Applications 3,7-dihydroxyphenoxazine (Amplex Red; Molecular Probes), producing resorufin, which gives rise to a red fluorescence (abs/em ∼ 570/585 nm). This substrate has only been employed for fluorometric detection of hydrogen peroxide [114, 117–119] and, to our knowledge, has never been used in β-Galactosidase immunohistochemistry. hydrolyzes the substrate di-β-galactopyranoside, producing fluorescein. This substrate has been used to identify the β-galactosidase gene [120, 121]. These fluorogenic substrates have not been reported in co-localization studies, although an eventual possibility for future application remains open. 1.4 QUADRUPLE IMMUNOSTAINING ON THE SAME SECTION Attempts to simultaneously identify four different antigens on tissue sections have been reported. However, the three methods described have drawbacks and cannot be generally used. 1.4.1 Quadruple Immunoenzymatic Staining Immunoenzymatic methods were the first to be performed for demonstrating four different hormones in the pituitary gland, where a triple–PAP staining was combined with an indirect method using β-galactosidase-labeled secondary antibodies [122]. In this technique, the first antigen was visualized using a monoclonal antibody and the PAP technique with DAB–cobalt as chromogen, giving a black-colored reaction product. This was followed by a second primary monoclonal antibody to the second antigen, which was detected using PAP and DAB, giving a 18
brown color. The third antigen was visualized by sequential incubations with two antibodies, first with a monoclonal antibody to the third antigen, detected with PAP and DAB for a short time to give a light brown color, followed by incubation with a polyclonal antibody raised to the same third antigen, demonstrated with βgalactosidase, which gives a turquoise-blue color. Thus, the third antigen was seen as green, due to the mixture of light brown and turquoise. The fourth antigen was detected by a polyclonal antibody, detected with β-galactosidase, which was seen as turquoise blue. This procedure has all the problems mentioned for double–PAP staining concerning evaluation of the mixed-color products and DAB interactions with the following staining steps (see Section 1.3.1.2.2), problems that are increased by the two additional staining sequences. 1.4.2 Single–PAP Combined with Triple Immunofluorescence Staining Øster et al. [123] described a quadruple immunostaining, achieved by first detecting one nuclear gene antigen using the PAP technique with DAB. This was followed by visualization of three cytoplasmic hormone antigens, using a triple immunofluorescence staining. A mixture of three primary antibodies raised in different animal species was detected by secondary antibodies conjugated with either a fluorophore or biotin. Because of the interferences caused by DAB (see Section 1.3.1.2.2), this method is useful if the antigen visualized with the PAP/DAB method is known to be localized in structures different than those antigens demonstrated with immunofluorescence techniques.
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens 1.4.3 Quadruple Immunofluorescence Staining A quadruple immunofluorescence method was performed using a mixture of primary antibodies raised in different animal species (sheep, mouse, monkey, rabbit and/or guinea pig), followed by incubation with fluorophore-conjugated secondary antibodies [103]. The fluorophores used were AMCA, FITC or Cy 2, Cy 3, and Cy 5, showing blue, green, yellow-green, and red fluorescence, respectively. Single filters specially built to select appropriate wavelengths were used. As mentioned in Section 1.3.2.3, Cy 3 usually exhibits a red fluorescence with the commercially available filters but with this specially constructed filter, the yellow region of the spectrum, where Cy 3 has its highest emission, was selected. Cy 5 is usually used in confocal microscopy because it emits light in the far-red region of the spectrum, which is detected with sensitive electronic devices; but by using specially designed filters and a microscope condenser, it was seen as red with a standard fluorescence microscope. With this method, four different antigens can be determined in the individual structures without any interference using four different single microscope filters. To evaluate co-localization, photographs taken with the four single filters had to be compared. Thus, the substances could be demonstrated at the same time but only sequencially by changing the different filters, to avoid color interferences, as the colocalization of red and green, seen as yellow, would be similar to the yellow color displayed by Cy 3 alone. Furthermore, to carry out this method, it is necessary to have both the microscope modifications and the appropriate filters, which are not commercially available.
1.5 CONCLUSIONS There are several methods to examine the co-localization of antigens in light microscopy. Single immunostaining on consecutive sections is a reliable method for co-localization studies of two antigens in relatively large structures such as cells, but not of thin structures such as nerve fibers. Double or triple immunostaining on the same section can be carried out with bright-field light microscopy; elution or non-elution techniques can be used, provided one is aware of the drawbacks. However, immunofluorescence techniques are the method of choice for co-localization studies of two or three antigens on the same section with light microscopy. If care is taken to avoid cross-reactivity of antibodies, there is no interference of the fluorophores in the individual structures, and there is no mixing of colors with appropriate microscope filters. Quadruple immunostaining on the same section has been attempted but cannot be currently used to demonstrate the four antigens at the same time due to its drawbacks.
1.6 PROTOCOLS I, II, III, IV, V, AND VI The following protocols are used by the author for specimens fixed in 10% buffered neutral formalin for 18 to 20 hr at room temperature (RT), followed by dehydration in graded ethanols and embedding in paraffin wax. Sections 5 µm thick are either attached to poly-L-lysine-coated glass slides or placed on positively charged glass slides (Superfrost+, Menzel, Germany). The primary antibodies are diluted 19
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Molecular Morphology in Human Tissues: Techniques and Applications in 1% bovine serum albumin (BSA) in phosphate buffer saline pH 7.4 (PBS), the secondary antibodies in PBS. All antibodies are applied at established dilutions. Descriptions of other methods are found in the references cited in the text. 1.6.1 Protocol I: Triple Immunofluorescence Staining with Primary Antibodies Raised in Different Animal Species, Using the Streptavidin–Biotin Complex (S-ABC) Method and Indirect Methods [74, 75] 1. Remove wax in xylene, hydrate through graded ethanols, and rinse in PBS. 2. Incubate with non-immune sera from the animal species producing the secondary antibodies, at a dilution of 1:10, 30 min at RT. Draw off sera. 3. Incubate with a cocktail of primary antibodies [one monoclonal + two polyclonal (e.g., anti-rabbit and antiguinea pig)], overnight at RT. 4. Rinse three times in PBS. 5. Incubate with biotinylated goat antiguinea pig IgG, 30 min at RT. 6. Rinse three times in PBS. 7. Incubate with a mixture of: fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit IgG + Texas Red (TXRD)-conjugated streptavidin + aminomethyl coumarin acetic acid (AMCA)-conjugated goat anti-mouse IgG, 30 min at RT. 8. Rinse three times in PBS. 9. Dry the slides except around the section and mount in an anti-fading nonfluorescent aqueous mounting medium. 10. Keep the slides in the dark at 4°C before examination with the fluorescence microscope. 20
1.6.2 Protocol II: Double Immunofluorescence Staining with Primary Antibodies Raised in Different Animal Species [76–78] Double immunofluorescence staining is performed in the same way as in Protocol I except for exclusion of one of the primary antibodies and corresponding secondary antibodies. 1.6.3 Protocol III: Double Immunofluorescence Staining with Primary Antibodies Raised in the Same Animal Species (e.g., Rabbit) Using Monovalent Fab Fragments of IgG [74] 1. & 2. As in Protocol I. 3. Incubate with the first primary antirabbit antibody, overnight at RT. 4. Rinse three times in PBS. 5. Incubate with monovalent FITCconjugated goat Fab anti-rabbit IgG, overnight at RT. 6. Rinse three times in PBS. 7. Incubate with the second primary anti-rabbit antibody, overnight at RT. 8. Rinse three times in PBS. 9. Incubate with biotinylated swine anti-rabbit IgG, 30 min at RT. 10. Rinse three times in PBS. 11. Incubate with TXRD-conjugated streptavidin, 30 min at RT. 12. Rinse three times in PBS and mount as in Protocol I. 1.6.4 Protocol IV: Double Immunofluorescence Staining with Primary Antibodies Raised in Different Animal Species Using the Catalyzed Reporter Deposition (CARD) Method and an Indirect Method [70–73] 1. & 2. As in Protocol I.
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens 3. Incubate with a cocktail of primary antibodies [e.g., one monoclonal (anti-mouse) giving a strong staining reaction + one polyclonal (anti-rabbit) giving a weak reaction], overnight at RT. 4. Rinse three times in PBS. 5. Incubate with biotinylated swine anti-rabbit IgG, 30 min at RT. 6. Rinse three times in PBS. 7. Incubate with horseradish peroxidaseconjugated streptavidin, 30 min at RT. 8. Rinse three times in PBS. 9. Incubate with biotinylated tyramide, at established time at RT. 10. Rinse three times in PBS. 11. Incubate with a mixture of TXRDconjugated streptavidin and FITCconjugated goat anti-mouse IgG, 30 min at RT. 12. Rinse three times in PBS and mount as in Protocol I. 1.6.5 Protocol V: Double Immunofluorescence Staining with Primary Antibodies Raised in the Same Animal Species (e.g., Rabbit) Using the Catalyzed Reporter Deposition (CARD) Method and an Indirect Method, with Paraformaldehyde Vapor Treatment [70, 75] 1. & 2. As in Protocol I. 3. Incubate with the primary antibody to be enhanced by the CARD method, overnight at RT. 4. to 10. As in Protocol IV. 11. Incubate with TXRD-conjugated streptavidin, 30 min at RT. 12. Rinse three times in PBS. 13. Dehydrate through graded ethanols to xylene, and let the sections dry.
14. Expose the sections to paraformaldehyde vapor, 4 hr at 80°C. 15. Rinse three times in PBS. 16. Incubate with 1% BSA in PBS, 15 min at RT. Draw off. 17. Incubate with the second primary antibody. 18. Rinse three times in PBS. 19. Incubate with FITC-conjugated goat anti-rabbit, 30 min at RT. 20. Rinse three times in PBS and mount as in Protocol I. 1.6.6 Protocol VI: Double Immunofluorescence Staining with Primary Antibodies Raised in the Same Animal Species (e.g., Rabbit) Using a Double Catalyzed Reporter Deposition (CARD) Method [73, 85] 1. & 2. As in Protocol I. 3. Incubate with the first primary antibody, overnight at RT. 4. to 10. As in Protocol IV. 11. Incubate with unlabeled avidin (100 mg/ml), overnight at RT. 12. Rinse three times in PBS. 13. Incubate with the second primary antibody, overnight at RT. 14. Rinse three times in PBS. 15. Incubate with biotinylated goat antirabbit, 30 min at RT. 16. Perform the enhancement with the CARD method as in Protocol IV, steps 6 through 10. 17. Incubate with FITC-conjugated streptavidin, 30 min at RT. 18. Rinse three times in PBS and mount as in Protocol I. Note: Alexa Fluor dyes 488, 594, and 350 can be used instead of FITC, TXRD, and AMCA, respectively.
21
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Molecular Morphology in Human Tissues: Techniques and Applications Acknowledgments
The experimental work was performed at the Department of Genetics and Pathology, University of Uppsala, Sweden, supported by grants from the Cancer Foundation and the Swedish Medical Research Council (Project No. 102). We are grateful to Professor Lars Grimelius for generous support and laboratory facilities.
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens 23. Gown, A.M. et al., Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures, J. Histochem. Cytochem., 34, 403, 1986. 24. Sakanaka, M. et al., A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining, J. Histochem. Cytochem., 36, 1091, 1988. 25. Cordell, J.L. et al., Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal antialkaline phosphatase (APAAP complexes), J. Histochem. Cytochem., 32, 219, 1984. 26. Malik, N.J. and Daymon, M.E., Improved double immunoenzymatic labeling using alkaline phosphatase and horseradish peroxidase, J. Clin. Pathol., 35, 1092, 1982. 27. Campbell, G.T. and Bhatnagar A.S., Simultaneous visualization by light microscopy of two pituitary hormones in a single tissue section using a combination of indirect immunohistochemical methods, J. Histochem. Cytochem., 24, 448, 1976. 28. Suffin, S.C. et al., Improvement of the glucose oxidase immunoenzyme technique, Am. J. Clin. Pathol., 71, 492, 1979. 29. Bondi, A. et al., The use of β-galactosidase as a tracer in immunohistochemistry, Histochemistry, 76, 153, 1982. 30. Lojda, Z., Indigogenic methods for glycosidases. II. An improved method for β-D-galactosidase and its application to localization studies of the enzyme in the intestine and in other tissues, Histochemie, 23, 266, 1970. 31. Gossrau, R., On the histochemical and microchemical demonstration of beta-galactosidase by means of 1-naphthyl-beta-galactopyranoside, Histochemie, 35, 199, 1973. 32. Larsson, L.-I., Peptide immunocytochemistry, Progress Histochem. Cytochem., 13, 1, 1981. 33. Farr, A.G. and Nakane, P.K., Immunohistochemistry with enzyme labeled antibodies: a brief overview, J. Immunol. Meth., 47, 129, 1981. 34. Pluzek, K.J. et al., A major advance for immunocytochemistry: enhanced polymer one-step staining (EPOS), J. Pathol., 169 (Suppl.), Abstr. 220, 1993. 35. Pastore, J.N. et al., A rapid immunoenzyme double labeling technique using EPOS reagents, J. Histotechnol., 18, 35, 1995. 36. Van der Loos, C.M., Naruko, T., and Becker, A.E., The use of enhanced polymer one-step staining reagents for immunoenzyme doublelabeling, Histochem. J., 28, 709, 1996.
37. Sternberger, L.A. et al., The unlabeled antibody–enzyme method of immunohistochemistry. Preparation and properties of soluble antigen–antibody complex (horseradish peroxidase–anti-horseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem., 18, 315, 1970. 38. Joseph, F.A. and Sternberger, L.A., The unlabeled antibody method. Contrasting color staining of β-lipotropin and ACTH-associated hypothalamic peptides without antibody removal, J. Histochem. Cytochem., 27, 1430, 1979. 39. Sternberger, L.A. and Joseph, F.A., The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal, J. Histochem. Cytochem., 29, 1424, 1979. 40. Lechago, J., Sun, N.C.J., and Weinstein, W.M., Simultaneous visualisation of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques, J. Histochem. Cytochem., 27, 1221, 1979. 41. Hsu, S.M., Raine, L., and Fanger, H., The use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled (PAP) procedures, J. Histochem. Cytochem., 29, 577, 1981. 42. Guesdon, J.L., Ternynck, T., and Avrameas, S., The use of avidin–biotin interaction in immunoenzymatic techniques, J. Histochem. Cytochem., 27, 1131, 1979. 43. Heras, A., Roach, C.M., and Key, M.E., Enhanced polymer detection system for immunocytochemistry, Mod. Pathol., 8, 165A, 1995. 44. Vyberg, M. and Nielsen, S., Dextran polymer conjugate two-step visualization system for immunohistochemistry, Appl. Immunohistochem., 6, 3, 1998. 45. Shi, S.-R. et al., Sensitivity and detection efficiency of a novel two-step detection system (PowerVision) for immunohistochemistry, Appl. Immunohistochem. Molec. Morphol., 7, 201, 1999. 46. Lehr, H.A. et al., Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis, J. Histochem. Cytochem., 47, 119, 1999. 47. Clark, C.A., Downs, E.C., and Primus, F.J., An unlabeled antibody method using glucose oxidase–antiglucose oxidase complexes: a sensitive alternative to immunoperoxidase for the detection of tissue antigens, J. Histochem. Cytochem., 30, 27, 1982. 48. Joseph, S.A. and Piekut, D.T., Dual immunostaining procedure demonstrating neurotransmitter and neuropeptide codistribution in the same section, Am. J. Anat., 175, 331, 1986.
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Molecular Morphology in Human Tissues: Techniques and Applications 49. Van der Loos, C.M. et al., Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabeled monoclonal antibodies, J. Immunol. Meth., 117, 45, 1989. 50. Gu, J. et al., Sequential use of the PAP and immunogold staining methods for the light microscopical double staining of tissue antigens, Regul. Pept., 1, 365, 1981. 51. Hacker, G.W. et al., Silver acetate autometalography: an alternative enhancement technique for immunogold-silver staining (IGSS) and silver amplification of gold, silver, mercury and zinc in tissues, J. Histotechnol., 11, 213, 1988. 52. Holgate, C.S. et al., Immunogold-silver staining — new method of immunostaining with enhanced sensitivity, J. Histochem. Cytochem., 31, 938, 1981. 53. Hainfeld, J.F. and Furuya, F.R., A 1.4 nm gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177, 1992. 54. Van den Brink, W.J. et al., Combined β-galactosidase and immunogold-silver staining for immunohistochemistry and DNA in situ hybridization, J. Histochem. Cytochem., 38, 325, 1990. 55. Krenács, T. and Krenács, L., Immunogold-silver staining (IGSS) for single and multiple antigen detection in archieved tissues following antigen retrieval, Cell Vision, 4, 387, 1997. 56. Gillitzer, R., Berger, R., and Moll, H., A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzynmatic labeling, J. Histochem. Cytochem., 38, 307, 1990. 57. Speel, E.J.M. et al,. A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase–Fast Red reaction, J. Histochem. Cytochem., 40, 1299, 1992. 58. Van der Loos, C.M. and Becker, A.E., Double epi-illumination microscopy with separate visualization of two antigens: a combination of epipolarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining, J. Histochem. Cytochem., 42, 289, 1994. 59. Roth, J., Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for the localization of single and multiple antigens in paraffin sections, J. Histochem. Cytochem., 30, 691, 1982.
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60. Pickel, V.M., Chan, J., and Milner, T.A., Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neural antigens, J. Histochem. Cytochem., 34, 707, 1986. 61. Larsson, L.-I. and Schwartz, T.W., Radioimmunocytochemistry — A novel immunocytochemical principle, J. Histochem. Cytochem., 25, 1140, 1977. 62. Hunt, S.P., Allanson, J., and Mantyh, P.W., Radioimmunocytochemistry, in Immunocytochemistry. Modern Methods and Applications, 2nd ed., Polak, J.M. and van Noorden, S., Eds., Wright, Bristol, 1986, 99. 63. Grino, M. and Zamora, A.J., An in situ hybridisation histochemistry technique allowing simultaneous visualization by the use of confocal microscopy of three cellular mRNA species in individual neurons, J. Histochem. Cytochem., 46, 753, 1998. 64. Henderson, D.C. and Smithyman, A.M., The simultaneous detection of two protein antigens in lymphoid tissues by combining immunofluorescence and autoradiography, J. Immunol. Meth., 6, 115, 1974. 65. Silverstein, A.M., Contrasting fluorescent labels for two antibodies, J. Histochem. Cytochem., 5, 94, 1957. 66. Coons, A.H. and Kaplan, M.H., Localization of antigen in tissue cells, J. Exp. Med., 91, 1, 1950. 67. Coons, A.H., Leduc, E.H., and Connolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit, J. Exp. Med., 102, 49, 1955. 68. Adams, J.C., Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains, J. Histochem. Cytochem., 40, 1457, 1992. 69. Bobrow, M.N. et al., Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays, J. Immunol. Meth., 125, 279,1989. 70. Portela-Gomes, G.M. et al,. Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract, Histochem. Cell Biol., 111, 49, 1999. 71. Portela-Gomes G.M. et al., Expression of the five somatostatin receptor subtypes in endocrine cells of the pancreas, Appl. Immunohistochem. Molec. Morphol., 8, 126, 2000.
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Immunostaining Techniques for Co-Localization of Multiple Peptide Antigens 72. Portela-Gomes, G.M. et al., PACAP is expressed in secretory granules of insulin and glucagon cells in human and rodent pancreas. Evidence for generation of cAMP compartments uncoupled from hormone release in diabetic islets, Regul. Pept., 113, 31, 2003. 73. Portela-Gomes, G.M., Lukinius, A., and Grimelius, L., Synaptic vesicle protein 2, a new neuroendocrine cell marker, Am. J. Pathol., 157, 1299, 2000. 74. Portela-Gomes, G.M. et al., Complex co-localization of chromogranins and neurohormones in the human gastrointestinal tract, J. Histochem. Cytochem., 45, 815, 1997. 75. Portela-Gomes, G.M. et al., Co-localization of neuroendocrine hormones in the human fetal pancreas, Eur. J. Endocrinol., 141, 525, 1999. 76. Portela-Gomes, G.M. and Höög, A., Insulin-like growth factor II in the human adult and foetal pancreas, J. Endocrinol., 165, 245, 2000. 77. Portela-Gomes, G.M. and Stridsberg, M., Selective processing of chromogranin A in the different islet cells in human pancreas, J. Histochem. Cytochem., 49, 483, 2001. 78. Portela-Gomes, G.M. and Stridsberg, M., Region-specific antibodies to chromogranin B display various immunostaining patterns in human endocrine pancreas, J. Histochem. Cytochem., 50, 1023, 2002. 79. Negoescu, A. et al., F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence, J. Histochem. Cytochem., 42, 433, 1994. 80. Wang, B.L. and Larsson, L.-I., Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining. Novel light and electron microscopical double and triple staining method employing primary antibodies from the same species, Histochemistry, 83, 47, 1985. 81. Hunyady, B. et al., Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining, J. Histochem. Cytochem., 44, 1353, 1996. 82. Jackerott, M. and Larsson, L.-I., Immunocytochemical localization of the NPY/PYY Y1 receptor in enteric neurons, endothelial cells, and endocrine-like cells of the rat intestinal tract, J. Histochem. Cytochem., 45, 1643, 1997. 83. Speel, E.J.M., Ramaekers, F.C.S., and Hopman, A.H.N., Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification, J. Histochem. Cytochem., 45, 1439, 1997.
84. Bobrow, M.N., Shaughnessy, K.J., and Litt, G.J., Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays, J. Immunol. Meth., 137, 103, 1991. 85. Portela-Gomes, G.M. and Abdel-Halim, S.M., Overexpression of Gs-proteins and adenylyl cyclase in normal and diabetic islets, Pancreas, 25, 176, 2002. 86. Erlandsen, S.L. et al., A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone, J. Histochem. Cytochem., 23, 666, 1975. 87. Mason, D.Y. and Woolston, R.E., Double immunoenzymatic labeling, in Technniques in Immunocytochemistry, Vol. 1, Bullock, G.R. and Petrusz, P., Eds., Academic Press, London, 1982, 135. 88. Landstorp, P.M. et al., Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5, Cytometry, 12, 723, 1991. 89. Johnson, G.D. et al., Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy, J. Immunol. Meth., 55, 231, 1982. 90. Steinbach, G., Characterization of fluorescein isothiocyanate: synthesis and testing methods for fluorescein isothiocyanate isomers, Acta Histochem., 50, 19, 1974. 91. Ernst, L.A. et al., Cyanine dye labeling reagents for sulphydryl groups, Cytometry, 10, 3, 1989. 92. Mujumdar, R.B. et al., Cyanine dye labeling reagents containing isothyocyanate groups, Cytometry, 10, 11, 1989. 93. Whitaker, J.E. et al., Fluorescent Rhodol derivatives: versatile, photostable labels and tracers, Anal. Biochem., 207, 267, 1992. 94. Sun, W.-C. et al., Synthesis of fluorinated fluoresceins, J. Org. Chem., 62, 6469, 1997. 95. Benchaib, M. et al., Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy, Histochem. Cell Biol., 106, 253, 1996. 96. Karolin, J. et al., Fluorescence and absorption spectroscopic properties of dipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipid membranes and proteins, J. Am. Chem. Soc., 116, 7801, 1994. 97. Lefevre, C. et al., Texas Red-X, new derivatives of sulforhodamine 101 and lissamine rhodamine B with improved labeling and fluorescence properties, Bioconjugate Chem., 7, 482, 1996.
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113. Mahmudi-Azer, S. et al., Inhibition of nonspecific binding of fluorescent-labeled antibodies to human eosinophils, J. Immunol. Meth., 21, 113, 1998. 114. Hauglhand, R.P. et al., Enzymatic Analysis Using Substrates That Yield Fluorescent Precipitates, U.S. Patent No. 5,316,906, 1994. 115. Tubbs, R.R. et al., Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH): a fluorescence-based assay enabling simultaneous visualization of gene amplification and encoded protein expression, J. Mol. Diagn., 2, 78, 2000. 116. Larison, K.D. et al., Use of a new fluorogenic phosphatase substrate in immunohistochemical applications, J. Histochem. Cytochem., 43, 77, 1995. 117. Mohanty, J.G. et al., A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine, J. Immunol. Meth., 28, 131, 1997. 118. Peus, D. et al., H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes, J. Invest. Dermatol., 110, 966, 1998. 119. Zhou, M. et al., A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases, Anal. Biochem., 253, 162, 1997. 120. Angelotti, T.P., Uhler, M.D., and Macdonald, R.L., Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique, J. Neurosci., 13, 1418, 1993. 121. Lin, S., Yang, S., and Hopkins, N., lacZ expression in germline transgenic zebrafish can be detected in living embryos, Dev. Biol., 161, 77, 1994. 122. Van Noorden, S. et al., Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies, J. Histochem. Cytochem., 34, 287, 1986. 123. Øster, A. et al., Rat endocrine pancreatic development in relation to two homeobox gene products (Pdx-1 and Nkx 6.1), J. Histochem. Cytochem., 46, 707, 1998.
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Multiple Staining in Molecular Morphology Chris M. van der Loos
2.1 INTRODUCTION During the past decade, the use of polyand monoclonal antibodies for in situ cell detection at the light microscopical level has developed into a powerful tool for both research and diagnostic purposes. A large number of technical approaches were developed by many investigators, as reviewed by Polak and Van Noorden [1] and Brandtzaeg [2]. Lately, commercially available immunohistochemical kit systems have also become available for single and double immunoenzyme staining. However, the visualization of multiple antigens in one tissue specimen using different enzyme chromogens is still believed to be restricted to “happy few” with “golden hands.” This belief seems justified by the fact that most multiple staining techniques are published as “applied papers,” describing a particular aim and sometimes including the preparation of enzyme and or hapten conjugates. This approach therefore cannot be regarded as “generally applicable” and “reproducible.” This chapter therefore focuses on double and triple immunoenzyme staining methods that can be fully carried out with commercially available reagents. This chapter offers a guide to the many different multiple staining methods and color com0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
binations, which usually frightens off the starter in this field. Furthermore, in Section 2.6, general protocols are provided that are easily adapted to detailed working procedures for many different, multiple staining applications. 2.1.1
Serial Sections
To complete, however, an immunophenotypic study, there is a regular demand for the detection of more than one antigen in a single tissue specimen. The traditional application of serial sections for this purpose [3] can certainly be regarded as reliable but extremely laborious. Moreover, (1) the serial sections must be very thin (1 to 2 mm) to avoid the risk that tiny structures or small cells may not be present in both sections; this requires special tissue fixation/embedding procedures and expensive microtomes. These procedures may also damage tissue antigens. (2) The “cellto-cell” comparison within homogeneous structured tissues (lymphoid tissue) is complicated due to the lack of tissue landmarks. Interesting co-localizations or cell-to-cell interactions will be missed easily. (3) Cytospin, cell smears, cell culture, and imprint specimens definitely cannot be studied in this way for obvious reasons. The
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Molecular Morphology in Human Tissues: Techniques and Applications performance of a reliable multiple staining at one tissue specimen is the best alternative to overcome these disadvantages. 2.1.2
Co-Localization: The Definition
Apart from staining two different cell types in a single tissue section (Plate 2.1{1, 5, and 6}), the observation of co-localization (i.e., the presence of two antigens in one cell) is one of the main reasons for the
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performance of double staining. When the two antigens are present in the same cellular compartment, co-localization will be marked by a mixed color: for example, the co-localization of two macrophage markers (Plate 2.1{3}) or the activation state of Tcells (Plate 2.2{7}). When the two antigens are present in different cellular compartments, co-localization will be observed as two different colors: for example, the nuclear staining of a proliferation marker within a cytokeratin positive cell or a cyto-
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Multiple Staining in Molecular Morphology plasmic and membrane marker (Plate 2.1{4}). It is obvious that, for proper identification of co-localization at this subcellular level, the pros and cons of the procedure of choice should be carefully considered. This survey provides the background necessary to perform adequate and reliable staining methods, together with practical notes that allow successful application of double staining methods.
2.1.3 Aims for Double and Triple Staining Multiple staining as an investigatory tool provides a variety of applications that can be incorporated in projects that require microscopical detail of tissue specimens: • Staining of two (or three) different cell types. Visualizing different cell types provides a direct overview of their
PLATE 2.1 (FIGURES 1–6) (Color Plate 2.1[Figures 1–6] follows page 106.) (1) Formalin-fixed and paraffin-embedded tissue section from an atherosclerotic lesion showing detail of the intima with mixed smooth muscle cells (brown) and foam-cell macrophages (red). The DakoCytomation EnVision Double Stain kit (based on sequential double staining concept) was applied, using first mouse anti-smooth muscle alpha-actin (clone 1A4) stained in brown (HRP – DakoCytomation DAB+) and, secondly, mouse anti-CD68 (clone PG-M1) in red (AP–DakoCytomation Fast Red). Note: This staining was performed without using the “Double Staining Blocking Step.” Nuclei are counterstained in blue with haematoxilin. (Original magnification ×50.) (2) Formalin-fixed and paraffin-embedded tissue section from cerebellum showing the localization of Neurofilament (NF) and glial fibrilaric acidic protein (GFAP). Double staining based on indirect/indirect concept with different animal species of mouse antibodies; anti-NF (clone 2F11) in turquoise (beta-GAL – X-gal) and anti-GFAP (rabbit) in red (AP – New Fuchsin). Note the glia cells showing co-localization by a purple-blue mixed color (arrowheads). Color mixing is due to the scavenging of NF products by glia cells as this specimen is from a patient with a neurodegenerative disease. (Original magnification ×50.) (3) Formalin-fixed and paraffin-embedded tissue section from an atherosclerotic lesion showing detail of the intima with different macrophage populations. Double staining based on indirect/indirect concept with two mouse primary antibodies of different subclasses; antimacrophage (IgM, clone HAM56) in red (HRP – Vector NovaRed) and anti-CD68 (IgG3, clone PGM1) in blue (AP – Vector Blue). Most macrophages express both antigens in their cytoplasm, resulting in a purple-blue mixed color. Only a few blue macrophages appeared to be PG-M1 single-positive. HAM56 single-red was only present at endothelial cells in the adventitia (insert). (Original magnification ×40.) (4) Acetone-fixed cryostat section from tonsil showing detail of the paracortical area with some activated macrophages (CD163+). Double staining based on indirect/direct concept with DakoCytomation Animal Research Kit for in vitro biotinylation of CD68 antibody. Anti-CD68 (clone EBM11) in blue (AP – Fast Blue BB) and anti-CD163 (clone Ber-MAC3) in red (HRP – AEC). A purple mixed color is absent because CD163 is staining the macrophage membrane, whereas CD68 is localized in the cytoplasm. (Original magnification ×40.) (5) Acetone-fixed cryostat section from an inflammatory abdominal aorta aneurysm segment, showing detail of the adventitia: T-cells (red) showing follicular organization adjacent to fibrotic areas with smooth muscle cells, muscular vessels (turquoise) and scattered macrophages (brown). Note the close contacts between the T-cells and “starry sky macrophages near the follicle center. Immunoenzyme triple staining with anti-smooth muscle cell α-actin (clone 1A4, mouse IgG2a) in turquoise (beta-GAL – X-gal), anti-CD3 (clone SK1), FITC-conjugated in red (AP – DakoCytomation Fast Red) and anti-CD68 (clone EBM11, mouse IgG1) in brown (HRP – DakoCytomation DAB+). Nuclei are counterstained in blue with haematoxilin. (Original magnification ×40.) (6) Acetone-fixed cryostat section from normal kidney showing glomeruli (g) with capillaries (red), tubili (t) marked by a collagen matrix (blue), and smooth muscle cells (turquoise) marking the media of larger vessels (v). Immunoenzyme triple staining with anti-smooth muscle cell α-actin (clone 1A4, mouse IgG2a) in turquoise (beta-GAL – X-gal), anti-collagen type III (clone HWD1.1, mouse IgG1) in blue (AP – Fast Blue BB), and anti-von Willebrand factor (rabbit) in red (HRP – AEC). (Original magnification ×20.)
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•
•
•
•
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localization in the tissue (Plate 2.1{1, 5, and 6}). For example, possible cellto-cell spatial contacts will be revealed (see Plate 2.1{5}). Multiple staining using primary antibodies a priori known to be present at/in these two cell types will result into two different colors; co-localization will not be present. Comparison of the staining patterns of a newly developed antibody with an antibody of a known cellular distribution. Double staining can result either in two differently colored cell types or in co-localization. For example, De Boer et al. [4] described the presence of co-stimulatory molecules such as CD28, CD70 at the macrophage subpopulation in atherosclerotic specimens. Comparison of two antibodies with possible similarities. The difference (or similarity) between the staining patterns of two commercially available monoclonal antibodies for a particular tissue type is not always clear from the data sheets. Double staining on the tissue of interest may provide information about this feature. The presence of co-localization marked by a mixed color is highly likely to occur (Plate 2.1{3}). Determination of the activation state of cells. Double staining can be used to demonstrate activated cells vs. nonactivated or resting cells in tissue specimens (Plate 2.1{4} and Plate 2.2{7}). For example, Hosono et al. [5] described the enumeration of T-cells vs. some T-cell activation markers. Comparison of immunohistochemistry and in situ hybridization. The combination of these two staining techniques on one specimen allows, for example, the investigation of the cellular phenotype of viral-infected cells
[6], or the comparison of a particular antigen at both mRNA and protein levels [7]. Saving tissue sections, or cell specimens. Choice of a large amount of primary antibodies may cause a shortage of material in the case of small biopsies. To fulfill these aims, there are two major approaches: (1) double immunofluorescence and (2) double immunoenzyme techniques. The clear and sharp localization of antigens with single and double immunofluorescence techniques [2] is, of course, beyond dispute. However, these techniques suffer from well-known drawbacks, including: • Quenching of the fluorescence signal at excitation • Fading of the fluorescence signal upon room-temperature storage of specimens • Occurrence of autofluorescence caused by formaldehyde fixation, thus preventing its application for retrospective studies [2, 8] Immunoenzymatic staining techniques, on the other hand, do not have these drawbacks. This chapter describes the use of enzymatic techniques for multiple staining. Multiple staining with two fluorochromes or gold particles of different sizes as well as exotic combinations of immunoenzyme and immunofluorescence techniques, are not further elaborated here. 2.1.4 The Demands for Successful Multiple Staining All multiple staining techniques, whether using gold particles of different sizes for immunoelectron microscopy, using different fluorochromes or enzymes, share some very important features for the successful performance of these staining techniques:
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PLATE 2.2 (FIGURE 7) (Color Plate 2.2 [Figure 7] follows page 106.) (7) Acetone-fixed cryostat section from tonsil showing follicle center (f ) and adjacent parafollicular area (p). Double staining of direct/direct concept with antiCD25 (clone ACT-1, FITC-conjugated) and anti-CD3 (clone SK1, PE-conjugated). Resting T-cells are single CD3+, activated T-cells are CD3+/CD25+ positive, and some macrophages/monocytes are single CD25+. (Original magnification ×20.) (7a) Bright-field image showing anti-CD25 (clone ACT-1) in red (HRP – AEC) and FITC-conjugated anti-CD3 (clone SK1) in blue (AP – Fast Blue BB). (7b) Same area as 7a showing the optical density in artificial colors after spectral analysis by Richard Levenson (CRI Inc., Woburn, MA). Note that activated T-cells showing CD3/CD25 co-localization can be recognized by mixed color much better in 7b than in the original bright-field image 7a. (7c/7d) Picture in 7b can be split into pure red (7c) and green (7d) using Adobe Photoshop software (Mountain View, CA).
1.
2.
Two (three) different immunohistochemical detection systems that: • Do not show any cross-reactivity • Preferably employ commercially available reagents Two (three) different visualization methods that: • Have to show optimal color contrast
• Allow for discrimination between a mixed color at sites of co-localization and the basic colors Although the remark to use “preferably commercially available reagents” seems quite trivial, one should realize that over the years many investigators solved the problem of cross-reacting reagents by producing their own conjugates: for example,
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Molecular Morphology in Human Tissues: Techniques and Applications the labeling of primary antibodies [9] and secondary antibodies [10]. Despite yielding excellent results, this can rarely be repeated by others and cannot be regarded as generally applicable. For this very reason, in this chapter we focus on reliable multiple staining methods that can be fully performed using commercially available reagents. Five double staining methods are shown in protocol format in Section 2.6, including suggestions for dilutions of second/third-step reagents.
staining procedures, including enzymatic activity visualization, performed next to each other. The main problem with this concept for double staining is the prevention of cross-reactions of immunoreagents between the first and second staining sequence. Numerous modifications with or without removal of the first set of reagents have been described, including. 1.
Furthermore, for the performance of immunoenzyme double staining, many different color combinations with different marker enzymes have been proposed [11]. In this chapter attention focuses on three color combinations, including the different staining efficiencies of the different chromogen systems. Moreover, suggestions are made for immunoenzyme triple staining using three different enzymatic activities.
2.2 DOUBLE STAINING CONCEPTS The double staining concepts described here haven proven to fulfill the major demands and can be performed with commercially available reagents. Detailed blueprints of five basic concepts for double staining, including suggestions for dilution of second/third-step reagents, are found in Section 2.6. In the schematic drawing of the double staining concepts, the figures between brackets refer to the subsequent incubation steps as found in the protocols in Section 2.6. 2.2.1
Sequential Techniques
Sequential double staining techniques involve two complete immunoenzyme 32
2.
Antibody elution step after the complete performance of the first staining sequence. For this purpose, either acidic (glycin-HCl buffer, pH 2.1) or high ionic strength (3 M ammonium isothiocyanate) solutions [12], oxidation with a permanganate solution [13], or electrophoresis [14] have been proposed. However, Tramu et al. [13] and Vandesande [14] both described how, despite the application of acidic or high ionic strength solutions, high-affinity primary antibodies may retain at their binding site. We have confirmed this observation with, for example, antismooth muscle α-actin, clone 1A4 and anti-macrophage CD68, clone PG-M1 (Van der Loos, personal observation). The use of DAB as chromogen for HRP activity in the first staining sequence results into an effectively sheltering reaction product, completely covering the first set of immunoreagents [15]. This sheltering effect is believed so effective that elution of the first set of reagents is not necessary. However, it cannot be excluded that the effective sheltering by the DAB end-product may hide the second antigen as well, especially if both antigens are in close proximity to each other [16]. Other HRP chromogens, such as AEC, TMB or AP, and GAL, do not have this sheltering characteris-
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Multiple Staining in Molecular Morphology tic. Effective sheltering will be also provided by the immunogold-silver staining (IGSS) end-product, covering the first set of reagents with silver precipitate [17]. Moreover, large poly-
meric structures such as StrepABComplex/HRP [17,18], DakoCytomation EPOS, and EnVision reagents [19], will also show sheltering effects by steric hindrance.
Tissue specimen with two different antigens/epitopes
Mouse antibody
Rabbit antibody
Goat antibody
Swine antibody
Streptavidin
HR P
Peroxidase enzyme label
AP
Alkaline phosphatase enzyme label
Biotin label F IT C
PE
Fluorescein isothiocyanate label Phycoerythrin label HR P
HR P
HR P HR P
HR P
HR P
DakoCytomation EnVision™/HRP or EPOS™/HRP reagent
Legend for Figures 2.1 through 2.7.
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Molecular Morphology in Human Tissues: Techniques and Applications 3.
The first set of reagents can be effectively removed by a heat-induced antigen retrieval procedure with citrate buffer (100 mM, pH 6.0) [2, 20]. This procedure is obviously applicable for paraffin sections only as cryostat sections will not survive this harsh treatment.
Sequential double staining is useful for the evaluation of two different cell populations or cell constituents. The DakoCytomation EnVision doublestaining system is based on polymer technology [21] and provides a user-friendly system for sequential double staining on both paraffin sections and cryostat sections. In our hands, the included “double staining blocking step” (pH 2.1) was of no use. The sheltering of the DAB reaction product after the first staining sequence as described by Sternberger and Joseph [15] appeared very effective indeed. Figure 2.1 represents a schematic drawing of this commercially available kit system corresponding with Protocol D in Section 2.6.4. Plate 2.1{1} shows a typical
example of this protocol applied with two mouse monoclonal antibodies. Because a sequential double staining technique always inherits the risk of spurious double-stained structures, this technique is less recommended for those instances where mixed-color products are expected at sites of co-localization. Moreover, to study co-localization by mixed colors, DAB should be avoided. The brown, black, or grayish DAB reaction product is difficult to combine with some other chromogen, still yielding an appropriate intermediate mixed color (Van der Loos, personal observation). 2.2.2 Simultaneous Technique: Direct/Direct In a direct/direct concept, both primary antibodies are conjugated differently. In its most basic format, two primary antibodies are directly conjugated with two different enzymes. In terms of commercial reagents, this situation is rarely available. However,
(5) Doublestaining blocking step or heat-induced antigen retrieval step
HR P
(4) Development of HRP activity with DAB
HR P
AP (9) Development of AP activity with Fast Red
HR P
AP
(3) EnVision™/HRP
HR P
(8) EnVision™/AP AP
HR P
(1) 1st Non-specific blocking step
AP (2)
antibody 1
(7) HR P
antibody 2
(6) 2nd Non-specific blocking step
AP
Figure 2.1 Sequential double staining using the DakoCytomation EnVision Doublestaining Kit System.
34
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Multiple Staining in Molecular Morphology instead of enzymes, the primary antibodies can be also conjugated with biotin (with a streptavidin reagent as second layer), or fluorochromes/haptens (with anti-fluorochrome or anti-hapten as second layer) [9, 11, 22–25]. The direct/direct double staining concept is completely independent of the primary antibody species, Ig isotype, or IgG subclass. In contrast with sequential double staining, time-saving antibody cocktails can be applied for simultaneous double staining concepts. The enzymatic activities are developed sequentially, after completing all antibody and detection steps. Most promising is a combination of two fluorochrome-conjugated primary antibodies as regularly available for Fluorescent Activated Cell Sorter applications, for example, fluorescein isothiocyanate (FITC) and phycoerhytrin (PE). Next, a second layer consisting of a goat anti-FITC and rabbit anti-PE antibodies and a finally a layer using a cocktail of swine and anti-goat Ig and swine anti-rabbit Ig conjugated with two different enzymes. In this way, the activated status (CD25, Interleukin 2-receptor) of T-cells (CD3) could be visualized (Plate
HR P
F IT C (1) Normal Swine Serum
(2)
2.2{7}). Figure 2.2 represents a schematic drawing of the direct/direct concept, corresponding with Protocol E in Section 2.6.5. 2.2.3 Simultaneous Technique: Indirect/Indirect Indirect/indirect double staining concepts are performed with two primary antibodies raised in different species [26, 27]. Timesaving antibody cocktails can be used and both enzymatic activities are developed last as described above with direct/direct double staining concepts. It is strongly recommended to apply secondary antibodies raised in the same host, to prevent unexpected crossreactions. In its most basic concept, indirect/indirect double staining is performed as schematically shown in Figure 2.3, corresponding with Protocol F in Section 2.6. More sensitivity is obtained by inserting a three-step streptavidin-biotin procedure for one of the primary antibodies or a two-step detection via EnVision (DakoCytomation) or PowerVision (ImmunoVision) (Figure 2.4). Plate 2.1{2} is an example of the indirect/indirect double staining concept with two primary antibodies of rabbit and mouse (4) Swine antigoat Ig/HRP
(3) Goat antiFITC
antibody 1/FITC, mouse
PE (2)
AP
(4) Swine antirabbit Ig/AP
(3) Rabbit antiPE
antibody 2/PE, mouse
Figure 2.2 Direct/direct double staining technique using two fluorochrome-conjugated primary antibodies.
35
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Molecular Morphology in Human Tissues: Techniques and Applications (3) Goat antimouse Ig/HRP
(3) Goat antirabbit Ig/AP
HR P
(2)
AP
(2)
antibody 1, mouse
antibody 2, rabbit
(1) Normal Goat Serum
Figure 2.3 Indirect/indirect double staining technique using two primary antibodies of different animal species (basic concept).
HRP
HR P
(3) Goat antirabbit/biotin
(4) Streptavidin/AP
AP
HRP (3) EnVision™ goat anti-mouse Ig/HRP
HR P (2)
antibody 1, mouse
(2)
antibody 2, rabbit
(1) Normal Goat Serum
Figure 2.4 Indirect/indirect double staining technique using two primary antibodies of different animal species (enhanced concept).
36
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Multiple Staining in Molecular Morphology origin showing the localization of neurofilament and glial fibrilaric acidic protein marking glia cells in the brain. An elegant variant on this indirect/indirect concept is with two mouse monoclonal primary antibodies of different Ig type or IgG isotype [28]. Specifically raised secondary antibodies will even distinguish between mouse IgG2a and IgG2b primary antibodies (Southern Biotechnology Associates). A schematic representation of this procedure is given in Figure 2.5, corresponding with Protocol G in Section 2.6.7. Plate 2.1{3} shows an example of the indirect/indirect concept with two primary antibodies of different mouse immunoglobulin isotypes IgG and IgM. 2.2.4 Multistep Techniques: Indirect/Direct The indirect/direct double staining concept is based on the application of one unlabeled primary antibody in combina-
tion with a directly conjugated second primary antibody. The conjugated antibody can be either enzyme labeled [24], biotinylated [29], FITC-conjugated [25], or a DakoCytomation EPOS™/HRP reagent [30]. Using the indirect/direct multistep procedure, reliable results can be obtained even if both primary antibodies are of the same species, Ig isotype, or IgG subclass. The multistep staining procedure starts with the unlabeled primary antibody, followed by appropriate detection steps, but without developing the enzymatic activity. Before incubation with the second directly labeled primary antibody, an incubation step with normal serum equal to the host of the primary antibodies is inserted. This blocking step prevents the second directly labeled primary antibody from cross-reacting with detection reagents involved with the first unlabeled primary antibody. In this way, this detection reagent for the first primary antibody is “saturated” by inert immunoglobulins present in the normal serum step. The indirect/direct double staining concept is very (4) Streptavidin/HRP
(3) Goat anti-mouse IgG3/AP
(3) Goat anti-mouse IgG2a/biotin
HR P
AP
(2)
antibody 1, mouse IgG3
(2)
antibody 2, mouse IgG2a
(1) Normal Goat Serum
Figure 2.5 Indirect/indirect double staining technique using two mouse primary antibodies of different IgG-isotype.
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Molecular Morphology in Human Tissues: Techniques and Applications flexible with respect to the applied color combination. The reagent list in Section 2.6 provides many combinations of reagents for this double staining procedure, yielding different color combinations. After completing all antibody and detection steps, the two enzymatic activities are developed sequentially. The indirect/direct double staining concept is also described by others [22, 31]. A schematic representation of the indirect/direct concept is given in Figure 2.6, corresponding with Protocol H in Section 2.6.8. The latest development with respect to the indirect/direct double staining procedure is the use of the DakoCytomation Animal Research Kit. This kit is primarily designed for background-free mouse-onmouse applications using in vitro biotinylation of a mouse antibody by a biotinylated goat anti-mouse Fab fragment. We have proven this kit also applicable for double
(4) Normal Mouse Serum
staining on human tissues using two unlabeled mouse primary antibodies [32]. It appeared that “truly” covalently biotinylated antibodies work just as well as “ARKitbiotinylated” antibodies in a multistep indirect/direct double staining procedure. Whenever the mouse IgG concentration is known from the data sheet, minute amounts of any commercially available mouse antibody can be readily and easily biotinylated. The DakoCytomation ARKulator (free download from http://us.dakocytomation.com) is a helpful tool for calculation of exact volumes of diluent, primary antibody (use concentration about equal with three-step streptavidin-biotin detection), “Biotinylation Reagent” and “Blocking Reagent.” A schematic representation of the multistep indirect/direct double staining concept applying an ARK-biotinylated antibody is shown in Figure 2.7 (see Protocol I in Section 2.6.9). Plate 2.1{4} shows an example of the indirect/direct concept
(7) Goat antirabbit Ig/HRP
(4) Streptavidin/AP
HR P
AP
(6) Rabbit antiFITC
(1) Normal Goat Serum
(3) Goat antimouse Ig/biotin (2)
antibody 1, mouse
F IT C
(5)
antibody 2/FITC, mouse
Figure 2.6 Indirect/direct double staining technique using one unlabeled mouse primary antibody and one FITC-conjugated antibody.
38
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Multiple Staining in Molecular Morphology (4) Normal Mouse Serum
HR P
HR P (6) Streptavidin/AP
HR P AP (3) EnVision™ goat anti-mouse Ig/HRP
HR P (2)
antibody 1, mouse
(5)
antibody 2, mouse-ARKbiotinylated
(1) Normal Goat Serum
Figure 2.7 Indirect/direct double staining technique using one unlabeled mouse primary antibody and one Animal Research Kit-biotinylated antibody.
with two unlabeled mouse IgG1-subclass primary antibodies. The ARK-biotinylation of mouse primary antibodies has also shown applicable with the direct/direct double staining concept. After ARK-biotinylation of a mouse primary antibody, a second directly FITCconjugated mouse primary antibody was added and incubated as cocktail. This type of double staining has also been successful on rat and mouse tissues. After the description of five different basic concepts for double staining, one might start to wonder which of those concepts is the best applicable in what situation. The answer to this question will be that it will be fully dependent on the combination of the primary antibodies. An elegant way to
select the best double staining method is described in Section 2.4 of this chapter. 2.3 MULTIPLE STAINING STRATEGY AND CONTROLS 2.3.1
General Strategy
An immunophenotypic study usually starts with selecting a number of antibody markers relevant to the question. Occasionally, pilot experiments must be carried out to find the optimal fixation and embedding procedure. In the case of retrospective studies with archival specimens involved, the applicability of the selected antibodies should be checked [33] and/or tissue pretreatment procedures should be tested [34]. For efficiency reasons, a strategy as shown below can be followed. 39
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Molecular Morphology in Human Tissues: Techniques and Applications 2.3.1.1
Single Staining
Prepare tissue sections from all tissue blocks to be tested, enough for all immunophenotyping experiments, including single staining, double staining, and triple staining. If possible, cut (semi-) serial sections and number them. Paraffin sections can be stored as ribbons, while cryostat sections, cell specimens, etc. can be airdried, and stored unfixed at –80°C (see Protocol A in Section 2.6.1, Protocol B in Section 2.6.2, and Protocol C in Section 2.6.3, depending on the type of specimen). After microscopic observation of all single immunostained slides, a small number of “investigation representative” cases are selected for double staining. 2.3.1.2
Double Staining
Sections in storage are used to perform the double staining experiments. A pilot double staining experiment is performed first, using only a few sections, but including all positive and negative controls. This first experiment allows an evaluation of whether comparable staining patterns are obtained as with the original single staining experiments. Moreover, in case colocalization of two antigens is expected, mixed-stained cell structures should be distinguished from both basic colors. For sequential double staining, a red-brown (plus hematoxilin counterstain) color combination is recommended; for other double staining methods, red-blue or turquoisered. See also Section 2.4 of this chapter. 2.3.1.3
Triple Staining
Only a few combinations of two double staining experiments with one common antibody marker can be selected for triple staining. Combinations can be composed of, for example, one rabbit primary antibody with two mouse primary antibodies of 40
different subclass (Plate 2.1{6}) or three mouse primary antibodies — one unlabeled (IgG1), one FITC-conjugated (IgG1), and one unlabeled (IgG2a) (Plate 2.1{5}). Van den Brink et al. [33] described a spectacular triple staining starting with the sequential technique, including DAB chromogen and subsequent heat-induced antigen retrieval for removal of immunoreagents [18] followed by indirect/indirect double staining based on different animal species. In our experience, triple staining results are mainly helpful in verifying and demonstrating features that have been already evaluated from double staining procedures. It is obvious that all remarks on prevention of cross-reactivity between detection reagents must be taken into account even more than for double staining procedures. The most successful triple staining results have been obtained with the marking of three different cellular constituents with no co-localization involved. In our hands, the best color combinations for triple staining are turquoise (ß-GAL – Xgal), blue (AP – N-AS-MX-P/Fast Blue BB), red (HRP – AEC) and turquoise (ßGAL – X-gal), red (AP – Fast Red), brown (HRP – DAB), developed in the given order. Plate 2.1{6 and 5, respectively} are examples of these two triple staining color combinations. As can been seen in Plate 2.1{5}, the turquoise-red-brown color combination also allows a nuclear counterstain with hematoxilin [35]. 2.3.2 Double Staining Control Experiments Apart from the usual experiment related controls, additional controls should be included in the first series of double staining experiments. These consist of two specimens that are treated with all incubation steps and detection procedures, but in
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Multiple Staining in Molecular Morphology which primary antibody 1 or 2, respectively, have been omitted [4, 30]. These specimens should be stained in parallel with the true double staining experiment. Although other marker enzymes and chromogens are used, both halves of the double staining must show similar staining patterns and staining intensity as compared with the original single staining specimens. Moreover, these extra controls may provide additional information regarding interspecies cross-reactivity and false-positivity due to endogenous enzyme activities or unwanted binding of reagents. In case there is any doubt whether or not structures are truly double-stained, it is recommended to perform experiments with a reversed color combination, or different color combination. The same applies to the problem of counting cells that present the mixed color, because the intermediate color may show variability in appearance.
2.4 SELECTING A CONCEPT, DETECTION SYSTEM, AND COLOR COMBINATION FOR DOUBLE STAINING 2.4.1 Selecting a Double Staining Concept The aim for a double staining method is usually formulated during evaluation of individual single-staining specimens. This implies that the choice of an appropriate double staining concept should be fully tailored to the characteristics of those primary antibodies applied for the original individual single-staining experiments. The flowchart (Table 2.1) for selection is based on the idea that the double staining method of choice should be as simple as possible. For example, commercially available conjugates
have preference over self-conjugated antibodies. When ending up with a “No” to the previous query, the most frequently encountered situation is a pair of two mouse monoclonal antibodies of IgG1 subclass. Alternatives include: • Search for a replacement antibody either raised in a different species or a monoclonal antibody of different isotype or IgG subclass. Be careful on this point; although two antibodies may look the same with respect to their description in data sheets, they may yield different staining patterns. For example, in Plate 2.1{3}, two antibodies have been applied, both described as “macrophage markers,” whereas obvious differences can be demonstrated by double staining. Therefore, always check the replacement antibody for its staining pattern compared with the original antibody, preferably with a double staining experiment. • Purify the immunoglobulin fraction from a large amount of one of the primary antibodies, and conjugate either with FITC, biotin, or a hapten. From our practical experience, it appeared that the 200 to 400 µg of immunoglobulin purification (Protein A method) needed for labeling, should be purified from an antibody solution at least containing a total amount of 500 to 700 µg of immunoglobulin. It should be stressed, however, that we have encountered several primary antibodies that did not resist this mild biotinylation procedure. Therefore, this option should be considered in the first place as expensive and, secondly, as uncertain regarding the performance of the final conjugate. • The problems with self-conjugation of a purified immunoglobulin as described above are circumvented by 41
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Molecular Morphology in Human Tissues: Techniques and Applications TABLE 2.1
Selecting the Simplest Double Staining Concept
Is a mixed color expected at sites of co -localization?
YES
NO
Apply a sequential double staining method, the DakoCytomation EnVision Doublestain System. Be aware of the possibility of falsely double - or single -stained cells. It is stressed that the use of control experiments for this type of double staining is essential.
YES
Apply an indirect/indirect (simultaneous) double staining method with suitable secondary reagents. To minimize interspecies crossreactivity, use secondary reagents raised in the same species.
A sequential double staining method is NOT recommended
Are the primary antibodies raised in different species?
NO with two unlabeled polyclonal antibodies
NO with two unlabeled monoclonal antibodies
Is there a difference between the immunoglobulin isotype or IgG subclass of the two monoclonal primary antibodies?
YES
Apply an indirect/indirect (simultaneous) double staining method with suitable anti-mouse immunoglobulin or IgG subclass specific secondary reagents. Preferably raised in the same species.
YES
Apply a direct/direct (simultaneous) double staining method. Second/third-step reagents are fully dependent on the choice of the label.
YES
Apply an indirect/direct (multistep) double staining method combining a directly conjugated antibody with an unlabeled antibody of the same species.
YES
Use the DakoCytomation ARKit for biotinylation of one of both mouse primary antibodies. Then apply an indirect/direct (multistep) double staining method.
YES
Apply a simple conjugation procedure using this small amount of purified immunoglobulin. For example FITC or a hapten. After successful conjugation, an indirect/direct (multistep) double staining method can be applied.
NO Are both primary antibodies commercially available as a conjugate (enzyme, hapten, biotin, or fluorochrome? NO Is one of both primary antibodies commercially available as a conjugate (enzyme, hapten, biotin, or fluorochrome? NO Is the specific mouse Ig concentration known from the data sheet? NO Is 200–400 µg of purified immunoglobulin available for conjugation of one primary antibody?
NO There are no further options for a reliable double staining procedure.
42
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Multiple Staining in Molecular Morphology the application of the DakoCytomation Animal Research Kit, which appeared also useful for simple biotinylation of mouse primary antibodies for human tissues [32]. See also Section 2.2 of this chapter. Of the three mentioned options here, the ARKbiotinylation is highly recommended. 2.4.2 Selecting Two Individual Detection Systems After selecting two non-cross-reacting immunoenzyme methods as a general concept for double staining, the definitive selection of both detection systems depends on some additional considerations, including: 1.
The sensitivity of both immunohistochemical techniques should be matched to the antigen density. In case of low expressing antigens/epitopes, highly sensitive/effective detection systems are needed for appropriate visualization. The final staining sensitivity/efficiency consists of two components: a. The detection system itself: current detection systems differ in their sensitivity/efficiency; for example, a traditional two-step procedure is less sensitive than a three-step detection procedure (PAP, APAAP, double-bridge APAAP, StrepABComplex methods) or a modern two-step procedure using polymers such as EnVision (DakoCytomation) or PowerVision (ImmunoVision). Inefficient detection methods may result in negative or weak staining [16]. Table 2.2 provides an overview of current detection methods in relation to their presumed sensitivity/efficiency according to our insights.
b. Some substrate/chromogen systems will produce a reaction product more efficiently than others [36]. Table 2.3 provides an overview of current enzymatic visualization methods in relation to their presumed sensitivity/efficiency according to our insights. Sometimes it is possible to adapt the dilution of the primary antibody to compensate for a higher or lower staining sensitivity/efficiency. The given dilutions in Table 2.3 are a guideline only. 2. It is obvious that whenever for a single staining a highly sensitive/efficient detection system must be employed, this should be maintained for double staining. All five double staining concepts here allow the use of the extremely sensitive tyramide amplification procedure (“TSA” by PerkinElmer; “CSA” by DakoCytomation). Protocol H in Section 2.6.8 shows an example of the indirect/direct multistep procedure with TSA included. 3. Apply a chromogen that fits to the antigen to be detected: In contrast to fluorescence and immunogold-silver staining (IGSS) techniques, reaction products after enzymatic activity visualization techniques are generally less sharply localized. Diffusion of the reaction product during the enzymatic reaction is the main cause of this problem, whereas the crystal size also plays a role in this. Hence, each chromogen system has its own characteristic with respect to the reaction product sharpness. As a consequence, thin fibrils, for example, should be detected with a chromogen showing a sharply localized end-product, rather than a diffusely localized reaction product such as Fast Blue or X-gal (Table 2.4). The localization sharpness of some reaction products containing diazonium 43
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Molecular Morphology in Human Tissues: Techniques and Applications TABLE 2.2
Relative Sensitivity/Efficiency of Immunohistochemical Detection Systems
Strong
High
Moderate 2
5-st TSA 1 5-st CSA 2
3-st StrepABC
3-st Strept /conj
3 -st: GAM + SAG/HRP
5-st APAAP
3-st via anti -DIG
3 -st via anti -FITC
2-st PowerVision 3
3-st APAAP
3-st PAP 1-st EPOS/HRP 4
2-st EnVision 4
Moderate 1
high
Weak 2-st indirect
low
Note: st = step. 1 TSA = Tyramide signal amplification, Perkin & Elmer product. 2 CSA = Catalized signal amplification, DakoCytomation product. 3 PowerVision is an Immuno Vision product. 4 EnVision and EPOS are DakoCytomation products.
TABLE 2.3
Relative Sensitivity/Efficiency of Some Substrates/Chromogen Systems Applicable for Multiple Staining IGSS
IGSS HRP
-epipol-
-bf- NovaRed
HRP
HRP
HRP HRP
TMB(?)
DAB+
AEC
AP
AP
DAB
AP
AP
AP
GAL
NBT/BCIP DAKOFastRed New Fuchsin FastRed TR FastBlue BB X-gal/ironcyan Vector Red (fluo) Vector BLUE Vector Red (bf)
high
low
primary antibody dilution:
1/200-500
1/100
1/50
1/20
1/10
Note: epipol- = epipolarization; bf- = bright field
salts (Fast Red, Fast Blue, Vector Red, and Vector Blue) can be improved by addition of 0.1% Tween 20 to the reaction mixture. The mechanism of Tween 20 is unclear.
44
2.4.3 Selecting Antibody Tracers and Color Combinations With the development of immunohistochemical double staining techniques, many tracer enzyme and color combinations have been exploited as described by several different authors [11]. It appears from our experience that a selection of enzyme tracer(s) and color combination(s) will depend largely on the aim of the study, the struc-
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Multiple Staining in Molecular Morphology TABLE 2.4
Relative Sharpness of Localization of Some Substrate/Chromogen Systems
IGS
DAB New Fuchsin AEC NBT/BCIP NovaRed TMB (?)
Fast Red TR Vector Red
crisp
ture of tissues and cell characteristics, the double staining method opted for, and personal preferences (e.g., color blindness). The use of one tracer enzyme, which is revealed histochemically in two different colors, can be applied with sequential double staining methods only [12]. However, the combination of two different enzymatic activities allows sequentially, as well as simultaneously performed double staining methods. As previously stressed, sequential double staining techniques can visualize two different cellular constituents only and are not recommended for revealing mixed colors at sites of co-localization. Of the many different color combinations that have been proposed for double staining, in my opinion there are only three successful ones. The characteristics of these color combinations are given below. 2.4.3.1
Brown-Red [37]
HRP activity in brown → DAB for visualization AP activity in red → Fast Red or New Fuchsin for visualization • Two distinct sharply localized reaction products (Table 2.4), showing a moderate/poor color contrast (Table 2.5).
Fast Blue BB Vector Blue
X-Gal
diffuse
• Less suitable for observing a mixed color at sites of co-localization, because of the lack of a well-defined mixed color [9]. • A nuclear counterstain in blue with hematoxilin is optional. • The Fast Red reaction product is soluble in alcohol and xylene. Mount aqueously. • The New Fuchsin reaction product is slightly soluble in alcohol and xylene. With high staining intensities organic mounting is no problem; but with low staining intensities, aqueous mounting is recommended. The best way of organic mounting is to completely dry the specimen after rinsing with distilled water and then immerse in xylene for mounting with an organic medium. The newly developed DakoCytomation Permanent Red (K0695) is a Fast Red that can be mounted organically. 2.4.3.2
Red-Blue [9]
HRP activity in red → AEC or Vector Nova Red for visualization AP activity in blue → Fast Blue BB or Vector Blue for visualization • Good color contrast; better than brown-red and brown-blue. 45
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Molecular Morphology in Human Tissues: Techniques and Applications • AEC or Nova Red provides a sharply localized reaction product; Fast Blue BB and Vector Blue have a more diffuse localization (Table 2.4). • Very suitable for observing a mixed color at sites of co-localization (purple-brown mixed color). • A nuclear counterstain with Methyl Green is not optional because the green nuclear stain fades away shortly after aqueous mounting. • Fast Blue BB and AEC reaction products are soluble in alcohol and xylene. Mount aqueously. The AEC reaction product may fade in time and/or after strong daylight exposure. Vector Blue and Nova Red can be mounted up organically using VectaMount (containing no alcohol or xylene). 2.4.3.3
Red-Turquoise [24]
AP activity in red → Fast Red or New Fuchsin for visualization GAL activity in turquoise → X-GAL/ ironcyanide for visualization • Very good color contrast; sometimes even better than red-blue. • New Fuchsin or Fast Red provides a sharply localized reaction product; X-GAL/ironcyanide is more diffusely localized (Table 2.4). • Suitable for observing a mixed color at sites of co-localization (purple-blue mixed color). • A nuclear counterstain with hematoxilin is optional, provided that no purple/blue mixed color at sites of colocalization will be present. • AP reaction product with Fast Red is soluble in alcohol and xylene. Mount aqueously. The newly developed DakoCytomation Permanent Red (K0695) is a Fast Red that can be mounted up organically. 46
• The X-GAL reaction product can withstand both aqueous and organic mounting; however, the AP reaction product with New Fuchsin is slightly soluble in alcohol. Mount either aqueously or organically (with some loss of AP reaction product). The best method of organic mounting is to completely dry the specimen after rinsing with distilled water and then immerse in xylene. • X-GAL can be replaced by Bluo-GAL [38], rendering a more bluish reaction product than with X-GAL. 2.4.4 Double Staining: Possibilities for Computerized Imaging of Two Individual Colors? From the color combinations described above, the turquoise-red color combination shows superior contrast to the human eye, based on the fact that these colors are almost opposing each other in the subtractive color circle. For this reason we have subjected this color combination to computerized imaging. Three procedures were tested: 1.
Application of two narrow bandpass filters close to the optimum absorbance wavelengths of X-GAL and New Fuchsin reaction products. The red filter was “dissolving” the red stained cells nearly completely, showing the turquoise stained cells only. However, the blue filter is not completely “dissolving” the turquoise stained smooth muscle cells. Setting of a threshold gray value for those turquoise cells resulted in the loss of weakly stained red cells [39]. Tested blue filters with other nearby wavelengths appeared not to be effective either. Other color combinations, such as HRP, DAB (brown/yellow)/AP, Fast Blue (blue) for this purpose, did not work because of a wide
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2.
3.
absorbance spectrum for the DAB chromogen, which almost overlaps the spectrum of the Fast Blue chromogen (Van der Loos, personal observation). Lehr et al. [40] described the use of relatively simple commercially available software (Photoshop version 4.0 or higher, Adobe Systems, Mountain View, CA) for cellular imaging. With the aid of this software, specific colors can be selected and the mean density and pixel number can be read. We have found that this new imaging method worked well for double stained specimens with the turquoise/red combination applied (Lehr et al. [39]). As with the previous method, the disadvantage of losing weakly stained cells no longer exists. Also, other color combinations (brown-blue, red-blue) may work with this procedure (Lehr and Van der Loos, personal observations). Spectral imaging is the most recent and powerful method to distinguish between two colored reaction products, even in those cases in which both colors look very similar to the human eye [41, 42]. Spectral imaging can be used to analyze, for example, a double staining specimen composed of HRP in red (AEC) and AP in blue (Fast Blue BB). First, the entire tissue specimen is analyzed pixel-by-pixel with respect to the spectral characteristics. This spectral cube data set is then electronically compared with the spectra of the pure reaction products of both individual colors. Finally, this yields a picture composed of artificial colors and called the OD (optical density) picture. Already this OD gives better and more information with respect to mixed colors by co-localization than the original specimen. On top of
that, both colors can be individually imaged now in black-and-white. This process is shown step-by-step in Plate 2.2{7a–d}. 2.5 ADDITIONAL PRACTICAL TIPS AND COMMON MISTAKES WITH DOUBLE STAINING 2.5.1
Which Antigen, Which Color?
The answer to this difficult question is highly dependent on tissue type, cellular composition, antigen/epitope distribution, and personal preferences. Recommendations include: • For the detection of mixed colors at sites of co-localization, start testing the red-blue combination composed of HRP (AEC or Vector NovaRed) and AP (Fast Blue BB or Vector Blue). • Two different cell populations can be demonstrated successfully applying the brown-red combination. Brownblue (composed of DAB-Fast Blue BB) [27] or brown-purple/blue (composed of DAB-NBT/BCIP) [43] combinations may serve as alternatives. • Regarding the antigen/epitope distribution, the following rules based on the natural color densities of the different reaction products may serve as an initial guide (see Table 2.5). Comments: • This guide is not clear for the redbrown combination because it consists of two, nearly equally dense colored reaction products. For a given aim, both red-brown and brown-red color combinations should be tested. • When the first double staining gives unsatisfactory results, the reverse color combination should always be tested. 47
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Molecular Morphology in Human Tissues: Techniques and Applications TABLE 2.5
Double Staining Selection Guide
red-turquoise Antigen present in majority of cells: → stain in weakest color
turquoise
Antigen present in minority of cells: → stain in darkest color
red
Spectacular differences between two reversed color combinations can be observed [11]. 2.5.2 1.
2.
48
Be Aware Of Endogenous AP activity. Endogenous AP activity occurs in acetone-fixed cryostat sections, for example, in vessels. Add levamisole for inhibiting all AP isoenzymes, except for AP activity in small intestine [44]. It is reported that too high concentrations of levamisole may inhibit the activity of the AP label as well [45]. Because endogenous AP activity does not survive the fixation/embedding procedure, levamisole can be omitted for formalinfixed paraffin sections. Endogenous peroxidase activity. Sometimes, regular blocking methods on cryostat sections may fail when applying 0.1% azide + 0.3% peroxide in washing buffer [46]. In such cases, also test the more efficient blocking method with methanol + 0.3% peroxide, but be aware of its deleterious effects on many antigens and epitopes. Apart from these two methods there is a rather time-consuming but very effective option to block endogenous peroxidase activity employing a glucose oxidase reaction [47].
brown-blue
red-blue
red-brown
brown
blue
red or brown
blue
red
red or brown
3.
Endogenous biotin. Many mammalian tissues contain biotin as a natural component. Hence, the use of (strept) avidin-biotin techniques carries the risk of intrinsic (strept)avidin binding activity. Indeed, this type of (unwanted) staining has been found abundantly in acetone-fixed cryostat sections of liver, kidney, and intestinal organs [48]. Furthermore, most epithelia, spleen, but also myocardial tissue contain endogenous biotin. In general, the more sensitive the used detection system, the more endogenous biotin will be found. After fixation in buffered formalin and paraffin embedding, most intrinsic (strept)avidin-binding activity is no longer present [49]. However, after heat-induced antigen retrieval [34], especially when using Tris/EDTA pH 9.0, the intrinsic (strept)avidin binding is also retrieved very effectively. In contrast, formalinfixed tissues subjected to heat-induced antigen retrieval using citrate pH 6.0 [50] do not show intrinsic (strept) avidin-binding activity. The best method for the suppression of intrinsic (strept)avidin binding activity is the subsequent application of 0.1% (strept)avidin and 0.01% d-biotin for 20 minutes each [48]. Based on this procedure, commercially available kit systems from different companies are
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4.
available (Vector, DakoCytomation). From personal observations it appears that heavy endogenous biotin is not always completely inhibited by the procedure mentioned above, nor by commercially available kit systems. As mentioned, sensitive non-biotin-based polymer staining techniques such as EPOS, EnVision (DakoCytomation), or PowerVision (LabVision) should be used to circumvent streptavidin-biotin staining methods. Brown tissue pigments. Melanin, iron pigment after hemorrhages, ceroid pigment (oxidized lipoproteins) in macrophages, and lipofuchsin in cardiac myocytes can be present. In such cases, avoid the use of DAB [11].
2.5.3 When the Staining Intensity of One of the Components in Double Staining Is Too Low Be aware of the fact that, once established, a primary antibody dilution for single staining performed with a three-step streptavidin-biotin complex technique and DAB or AEC as peroxidase chromogen, this primary antibody dilution needs to be adapted when performing double staining with other detection systems or chromogens. In particular, GAL activity revealed with X-GAL and AP activity revealed with Fast Blue BB reveal a rather low efficiency/sensitivity as compared with, for example, NBT/BCIP, AEC, or DAB substrates (Table 2.3) [36]. This lower efficiency can be sometimes compensated using either a 2 to 4 times higher concentration of the primary antibody, a more sensitive detection method (Table 2.2), and/or overnight incubation at 4°C instead of 60 min at room temperature.
2.5.4 Order of Enzymatic Development For the sequential double staining technique, it is strictly necessary that the HRP activity using DAB as chromogen is developed first. Unlike the DAB reaction product, AEC or AP and GAL reaction products do not have the same potential for sheltering the first set of reagents [6]. For the simultaneous or multistep double staining techniques applying HRP and AP as enzyme labels, AP activity is generally developed first, and after a brief wash, HRP as second. It is thought that the peroxide in the HRP visualization medium may damage the AP activity [37]. Maintaining “good laboratory practice” we usually keep to this order of development. However, we have observed that AP reaction components also influence the HRP reaction. It appears that the shorter the AP reaction takes, the stronger the HRP reaction will be (Van der Loos, unpublished observation). If AP-first, HRP-second is really hampering an adequate observation of weak HRP activity, a reversed development procedure may be performed without any severe problems concerning the AP staining intensity (Van der Loos, unpublished observation). More or less the same accounts for the ß-GAL/AP combination. There is no real preference as to which of the substrates should be used first. However, ß-GAL is strongly influenced by HRP activity (Van der Loos, unpublished observations). Again, for reasons of “good laboratory practice” ß-GAL activity is therefore always developed first, AP second. For triple staining combinations with have used the order of ß-GAL (X-gal) – AP (Fast Blue BB) – HRP (AEC) (Plate 2.1{6}) and ß-GAL (Xgal) – AP (Fast Red) – HRP (DAB) – hematoxilin nuclear counterstain (Plate 2.1{5}). 49
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Molecular Morphology in Human Tissues: Techniques and Applications 2.5.5 How to Get Started: Ten Steps to Successful Double Staining 1.
2.
3. 4.
5.
6.
7.
50
Cut (semi)serial sections and perform single immunostaining; keep remaining sections in stock for double staining. Determine the characteristics of your primary antibodies (species, Ig isotype, IgG subclass) and check for the possibility of commercially available conjugates (biotin, FITC, phycoerythrin, DakoCytomation EPOS/HRP). Use flowchart (Table 2.1) to find the easiest and simplest double staining method. When ending up with two unlabeled, unconjugated mouse monoclonal antibodies of the same isotype/subclass, consider the use of one alternative primary antibody with different characteristics. Furthermore, the DakoCytomation Animal Research Kit can be employed for in vitro biotinylation of one or both mouse primaries. Select a color combination. During the observation of the single-staining specimens, try to imagine what your double staining would look like in a certain color combination. Keep in mind that some reaction products are quite diffusely localized and do not fit with very small structures (Table 2.4). Most of the time, your imagination will be similar to the recommendations given in Table 2.5. The red-blue color combination as obtained from HRP (AEC or Vector NovaRed) and AP (Fast Blue BB or Vector Blue) is a very good combination for starters. Adapt the dilution of the primary antibodies to the sensitivity/efficiency of the applied detection and chromogen system (Tables 2.2 and 2.3). Write a full double staining protocol, including a schematic drawing.
Check both protocol and scheme step-by-step for possible interspecies cross-reactivity. 8. Perform your double staining and compare with the original single staining specimens. Observe the development of the enzymatic activities under the microscope at low magnification. Too short (not reproducible) and too long (may influence following enzyme activity) should be avoided. Include a control experiment: two half-double staining experiments, with omission of one or both primary antibodies. 9. At this stage, do not handle too many slides in one experiment, especially when testing more than one antibody combination. 10. If necessary, perform the double staining in a reversed color combination. 2.5.6 Restrictions for the Visualization of a Mixed Color at Sites of Co-Localization Using Immunoenzyme Double Staining Techniques The main restriction for observing a mixed color at sites of co-localization using bright-field microscopy is the situation wherein the two antigens under study are present in highly varying amounts [2]. A subtle mixed color will be missed easily. In those situations, classical double immunofluorescence or the combination of an immunogold-silver staining (IGSS) technique with a (non-fading) fluorescent alkaline phosphate staining method should be considered [51]. With double staining on formalin-fixed and paraffin-embedded tissue sections, there might be a limitation with respect to the tissue pretreatment methods. Some
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Multiple Staining in Molecular Morphology antigens require, for example, pepsin digestion as pretreatment (e.g., cytokeratins, endothelial markers, etc.), whereas most other antigens require (or are not hampered by) heat-induced antigen retrieval using citrate pH 6.0 or Tris-EDTA pH 9.0 treatment. It is obvious that two antigens cannot be combined successfully for double staining on one tissue section.
2.6 PROTOCOLS AND TECHNICAL NOTES 2.6.1 Protocol A for General Immunohistochemistry - Paraffin Sections 1. 2. 3. 4.
5.
6.
Mount 4- to 6-µm-thick paraffin sections on adhesive-coated microscopic glass. Dry 1 hr at 60°C or overnight at 37°C. De-paraffinize in xylene, and rehydrate section in graded alcohols. Block endogenous peroxidase activity using 0.3% hydrogen peroxide in methanol for 20 min at room temperature [52]. Rinse with running tap water. Treatment with pepsin, trypsin, antigen retrieval, depending on the antigen to be detected (pepsin: 0.25% pepsin [Sigma P7000] in 10 mM HCl (15 min, 37°C), trypsin: 0.1% trypsin (Sigma T8128) in 50 mM Tris-HCl pH 7.8 (10 min, 37°C); antigen retrieval citrate buffer pH 6.0: 10 mM citric acid, adjusted with NaOH to pH 6.0 (15 min 100°C, 10 min cool-down); Tris-EDTA 9.0: 10 mM Tris + 1 mM EDTA, adjusted with HCl to pH 9.0 (15 min 100°C, 10 min cool-down)) Rinse with running tap water.
7.
Dry carefully around tissue section and encircle with water-repellant rim. 8. Rinse with washing buffer. 9. Perform the immunohistochemical procedure, including enzyme activity detection. 10. Wash with running tap water for 5 min. 11. Coverslip with an appropriate mounting medium. Comment: Primary antibodies are incubated either 30 to 60 min at room temperature or overnight at 4°C in a humidified chamber. Room temperature should be applied if no temperature is indicated. Note: In the following double staining protocols, Tris/HCl buffered saline (TBS) or phosphate buffered saline (PBS) is used for washing between the subsequent incubation steps (3 × 5 min). This washing step is omitted from the protocols. 2.6.2 Protocol B for General Immunohistochemistry: Cryostat Sections 1. 2. 3.
4. 5. 6. 7.
Mount 4- to 6-µm cryostat sections on coated microscopic slide. Air dry for at least 1 to 2 hr, preferably overnight under a fan. Wrap in Parafilm foil “face-to-face” (use small carton strips at the glass edges) and store dry in a closed box at –80°C or go to step 5. Make sure that Parafilm-foil packed tissue specimens reach room temperature before unwrapping. Fix in acetone (p.a. quality) for 10 min at 4°C. Air-dry briefly again (2 min). Encircle the tissue sections with a water-repellant rim option: Apply extra fixation with Zamboni fixative (1 min at room temperature) and 51
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Molecular Morphology in Human Tissues: Techniques and Applications rinse with washing buffer (3 × 2 min). 8. Block endogenous peroxidase activity using sodium azide (0.1%) + hydrogen peroxide (0.3%) in washing buffer for 20 min at room temperature [46]. 9. Rinse with washing buffer (3 × 2 min). 10. Perform immunohistochemical procedure, including enzyme activity detection. Option: Post-fix with 4% buffered formalin for 5 min. 11. Wash with running tap water for 5 min. 12. Coverslip with an appropriate mounting medium. 2.6.3 Protocol C for General Immunohistochemistry: Staining of Intracellular Antigens with Intact Cells (Cell Preparations, Cytospins, etc.) Note: For staining of membrane antigens with intact cells, follow Protocol B in Section 2.6.2 as for cryostat sections. 1.
Wash cell suspension three times with PBS without BSA. Concentration: ±2 × 10−6 cells/ml. 2. Soak 12-well Adhesion Slides in PBS. (For example, ER-302 coated with Adcell from Erie Scientific, Portsmouth, NH). 3. Add 10-µl cell suspension to each Reaction Field. Allow cells to adhere overnight at 4°C in a humidified incubation box. For most applications, slides can be kept this way for several days. 4. Fix cells with fresh 4% PFA in PBS (5 min, RT). Wash with PBS (3 × 2 min). Option: Rinse slides with distilled water and allow to dry under a ventilator. Wrap in Parafilm foil “face-to-
52
5. 6.
7.
8. 9.
face” (use little carton strips at the glass edges) and store in a box at –80°C. Allow to reach RT before unwrapping. Use 0.1% saponin (Sigma S7000) in all further steps [53]. Block endogenous peroxidase activity using 50 mg Na-azide + 500 µl hydrogen peroxide (30%) + 500 µl saponin (10%) in 50 ml TBS for 20 min RT [46]. Rinse three times with TBS. Perform immunohistochemical procedure, including enzyme activity detection. Option: Post-fix with 4% buffered formalin for 5 min. Wash with running tap water for 5 min. Coverslip with an appropriate mounting medium.
2.6.4 Protocol D for Sequential Immunoenzyme Double Staining Good results have been obtained using the newly designed DakoCytomation Doublestain System based on EnVision technology (K1395). (See Figure 2.1 for a schematic representation of this procedure.) 1. Serum-free protein blocking step, for 15 min. 2. Mouse or rabbit antibody 1, overnight at 4°C, or 30 to 60 min at room temperature. 3. EnVision/HRP, Goat anti-mouse/ Goat anti-rabbit, 30 min. 4. Development of HRP activity with DAB+ in brown, 2 to 10 min. 5. Elution step with Double Staining Block, 30 min. Alternatively, boiling for 5 min in citrate buffer pH 6.0 [20]. Wash with distilled water, 5 min. Non-specific binding blocking step, 5 min.
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Multiple Staining in Molecular Morphology 6. Serum-free protein blocking step, 15 min. 7. Mouse or rabbit antibody 2, overnight at 4°C, or 30 to 60 min at room temperature. 8. EnVision/AP, Goat anti-mouse/ Goat anti-rabbit, 60 min. 9. Development of AP activity detection with Fast Red in red, 5 to 30 min. Comment: The commercially available kit is a user-friendly system for the evaluation of two different cell populations, but is less recommended for those instances where mixed-color products are expected at sites of co-localization. 2.6.5 Protocol E for Simultaneous Immunoenzyme Double Staining: Direct/Direct Concept
ent species, it is recommended to apply reagents raised in the same host. Example with two fluorochromes: see Figure 2.2 for schematic representation. 1. Serum-free protein blocking step or Normal swine serum (1:10), for 15 min. 2. MAb1/FITC-conjugate + MAb2/PEconjugate, for 60 min. 3. Goat anti-FITC (1:400) + Rabbit anti-PE (1:200), for 15 min. 4. Swine anti-goat Ig/HRP (1:50) + Swine anti-rabbit Ig/AP (1:20), for 30 min. 5. AP activity detection in blue (Vector Blue). HRP activity detection in red (AEC or Vector NovaRed).
This protocol is based on two directly labeled primary monoclonal antibodies [9].
2.6.6 Protocol F for Simultaneous Immunoenzyme Double Staining: Indirect/Indirect (Concept 1)
1. Serum-free protein blocking step, for 15 min. 2. MAb 1/conjugate 1 + MAb 2/conjugate 2 (“cocktail”), overnight at 4°C, or 60 min at room temperature. Further steps are primary antibody-label dependent and need to be treated as usual practice. Then: 3. First enzyme activity detection. 4. Second enzyme activity detection.
This protocol is based on two unlabeled primary antibodies of rabbit and mouse origin [26, 27].
Comment 1: The markers involved with directly labeled primary antibodies can be either enzymes, biotin (with a streptavidin reagent as second layer), or fluorochromes/ haptens (with anti-fluorochrome or antihapten as second layer). DakoCytomation EPOS/HRP reagents can be regarded as directly HRP-conjugated primary antibodies [19, 30], and can therefore be incorporated into this type of protocol. Comment 2: To prevent cross-reaction between second-step reagents from differ-
1. Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min. 2. Mouse antibody 1 + Rabbit antibody 2 (“cocktail”), overnight at 4°C, or 60 min at room temperature. 3. GAM-enzyme I conjugated + GAR-enzyme II conjugated (“cocktail”), for 30 min. 4. First enzyme activity detection. 5. Second enzyme activity detection. For more sensitivity, one biotinylated second-step reagent (Step 3) can be incorporated into this protocol. This step is followed by an appropriate third-step streptavidin- or anti-biotin reagent. Furthermore, polymer techniques such as EnVision or PowerVision can be used. See 53
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Molecular Morphology in Human Tissues: Techniques and Applications Figure 2.3 for a schematic representation of this three-step procedure: 1. Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min. 2. Mouse antibody 1 + Rabbit antibody 2 (“cocktail”), overnight at 4°C, or 60 min at room temperature. 3. GAM-EnVision+/HRP (undiluted) + GAR/biotin (1:400) (“cocktail”), for 30 min. 4. Streptavidin/AP (1:100), for 30 min. 5. AP activity detection in blue (Fast Blue BB or Vector Blue). 6. HRP activity detection in red (AEC or Vector NovaRed). or: 1. Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min. 2. Mouse antibody 1 + Rabbit antibody 2 (“cocktail”), overnight at 4°C, or 60 min at room temperature. 3. GAM-EnVision+/HRP (undiluted), for 30 min. 4. GAR-PowerVision/AP (undiluted), for 30 min. 5. AP activity detection in blue (Fast Blue BB or Vector Blue). 6. HRP activity detection in red (AEC or Vector NovaRed). 2.6.7 Protocol G for Simultaneous Immunoenzyme Double Staining:– Indirect/Indirect (Concept 2) This protocol is based on two unlabeled primary mouse monoclonal antibodies of different Ig subtype or IgG isotype [11, 28]. 1. Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min.
54
2. Mouse antibody 1 (Ig isotype or IgG subclass X) + Mouse antibody 2 (Ig isotype or IgG subclass Y) (“cocktail”), overnight at 4°C, or 60 min at room temperature. 3. GAM-IgX enzyme I conjugated + GAM-IgY-enzyme II conjugated (“cocktail”), for 30 min. 4. First enzyme activity detection. 5. Second enzyme activity detection. Comment: As with the previous indirect/indirect concept, more sensitivity for at least one primary antibody can be obtained with a biotinylated second-step reagent (Step 3). This step should be followed by an appropriate third-step streptavidin-reagent (Figure 2.5): 1. Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min. 2. Mouse antibody 1 (IgG3) + Mouse antibody 2 (Ig2a) (“cocktail”), overnight at 4°C, or 60 min at room temperature. 3. GAM-IgG3/AP (1:20) + GAM-IgG2a/ biotin (1:100) (“cocktail”), for 30 min. 4. Strep/HRP (1:400), for 30 min. 5. AP activity detection in blue (Fast Blue BB or Vector Blue). 6. HRP activity detection in red (AEC or Vector NovaRed). 2.6.8 Protocol H for Multistep Immunoenzyme Double Staining: Indirect/Direct Concept This protocol is based on one unlabeled monoclonal antibody, one FITC-conjugated [25]. See Figure 2.6 for schematic representation of this procedure. 1. Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min.
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Multiple Staining in Molecular Morphology 2. 3. 4. 5. 6. 7. 8. 9.
Mouse MAb 1, overnight at 4°C, or 60 min at room temperature. GAM/biotin (1:200), for 30 min. Normal mouse serum (1:10) + streptavidin enzyme I reagent, for 30 min. Mouse MAb 2/FITC-conjugated, overnight at 4°C, or 60 min at room temperature. Rabbit anti-FITC (1:1000), for 15 min. GAR-enzyme II conjugated, for 30 min. First enzyme activity detection. Second enzyme activity detection. Alternatives include:
Three-step alkaline phosphatase anti-alkaline phosphatase (APAAP) detection of MAb 1, avoiding streptavidin/biotin interaction: 1. GAM (1:25), for 30 min. 2. APAAP complex, mouse (1:100), for 30 min. 3. Normal Mouse Serum (1:10), for 15 min. Continue with Step 5 of the above protocol. Two-step peroxidase detection of MAb 1, avoiding streptavidin/biotin interaction: 1. GAM-EnVision+™/HRP (undiluted), for 30 min. 2. Normal Mouse Serum (1:10), for 15 min. Continue with Step 6 of the above protocol. Comment: Apart from directly FITClabeled primary antibodies as frequently used for the Fluorescein Activated Cell Sorter, the Texas Red-, Rhodamine-, AMCA-, or phycoerythrine-labeled antibodies can also be applied for this double staining technique by inserting an appropriate Step 6 reagent. Also, biotinylated primary antibodies can be
applied at Step 5 [29], followed by a streptavidin or anti-biotin reagent. Like the DakoCytomation EnVision products, the Enhanced Polymer One-Step (EPOS) staining products basically consist of a dextran polymer backbone. With EPOS products, this backbone is labeled with a variety of primary antibodies (DakoCytomation U-class reagents) and HRP enzymes. EPOS products can be regarded as directly labeled primary antibodies and can therefore be applied in this double staining concept [30]. One of the most sensitive multistep indirect/direct double staining procedures works as follows, with biotinylated tyramides (e.g., as contained in the DakoCytomation “CSA” kits or the Perkin-Elmer “TSA” kits): 1. Serum-free protein blocking step or Normal Goat Serum (1:10), at 15 min. 2. Mouse MAb 1, overnight at 4°C, or 60 min at room temperature. 3. GAM/biotin (1:1000), for 30 min. 4. Streptavidin/HRP (1:200), for 30 min. 5. TSA/biotin reagent (1:200 in amplification diluent) (biotin-labeled tyramides), for 10 min. 6. Streptavidin/HRP (1:100), for 30 min. 7. Normal Mouse Serum (1:10), 15 min. 8. Mouse MAb 2/FITC-conjugated, overnight at 4°C, or 60 min at room temperature. 9. Rabbit anti-FITC (1:1000), for 15 min. 10. GAR-enzyme II conjugated, for 30 min. 11. First enzyme (HRP) activity detection. 12. Second enzyme activity detection.
55
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Molecular Morphology in Human Tissues: Techniques and Applications 2.6.9 Protocol I for Multistep Immunoenzyme Double Staining Using the DakoCytomation Animal Research Kit for In Vitro Biotinylation: Indirect/Direct Concept
4. 5. 6. 7. 8.
Normal Mouse Serum (1:10), for 15 min. Mouse MAb 2/ARK-biotinylated, overnight at 4°C, or 60 min at room temperature. Streptavidin/AP (1:100), for 30 min. First enzyme activity detection. Second enzyme activity detection.
This protocol is based on one unlabeled monoclonal antibody, one in vitro ARK-biotinylated antibody [32). See Figure 2.7 for a schematic representation of this procedure.
2.6.11 Non-Commercial Visualization Systems
1.
2.6.11.1 Protocol for HRP and AEC, modified after Graham et al. [54]
2. 3.
Serum-free protein blocking step or Normal Goat Serum (1:10), for 15 min. Mouse MAb, overnight at 4°C, or 60 min at room temperature. GAM, EnVision+/HRP (undiluted), for 30 min.
Stock solution: 25 mg 3-amino-9-ethylcarbazole (AEC) (Sigma A5754) dissolved in 2.5 ml N,N-dimethylformamide. Store in dark at +4°C. Mix 2.5 ml AEC stock solution with Na-acetate buffer (50 mM, pH 5.2); filter directly and add 20 µl
2.6.10 Suggested Immunoreagent List Reagent APAAP complex, mouse ARKit EnVision Doublestain System Goat anti-FITC Goat anti-mouse Ig Goat anti-mouse Ig/AP Goat anti-mouse Ig/biotin Goat anti-mouse Ig/GAL Goat anti-mouse Ig/HRP Goat anti-mouse, EnVision+/HRP Goat anti-mouse IgM/HRP Goat anti-mouse IgM/biotin Goat anti-mouse IgM/AP Goat anti-mouse IgG1/HRP Goat anti-mouse IgG1/biotin Goat anti-mouse IgG1/AP Goat anti-mouse IgG2a/HRP Goat anti-mouse IgG2a/biotin Goat anti-mouse IgG2a/AP Goat anti-mouse IgG2b/HRP Goat anti-mouse IgG2b/biotin Goat anti-mouse IgG2b/AP Goat anti-mouse IgG3/biotin Goat anti-mouse IgG3/AP Goat anti-rabbit Ig/AP Goat anti-rabbit Ig/biotin
Vendor and Code DakoCyto D0651 DakoCyto K3954/3955 DakoCyto K1395 BioGenesis 4510-7204 DakoCyto Z0420 DakoCyto D0486 DakoCyto E0433 SBA 4010-06 DakoCyto P0447 DakoCyto K4000/4001 SBA 1020-05 SBA 1020-08 SBA 1020-04 SBA 1070-05 SBA 1070-08 SBA 1070-04 SBA 1080-05 SBA 1080-08 SBA 1080-04 SBA 1090-05 SBA 1090-08 SBA 1090-04 SBA 1100-08 SBA 1100-04 DakoCyto D0487 DakoCyto E0432
Dilution
Time/Temp.
1:100
30 min, RT
1:400 1:25 1:20 1:200 1:10 1:50 undiluted 1:50 1:100 1:20 1:50 1:100 1:20 1:50 1:100 1:20 1:50 1:100 1:20 1:100 1:20 1:20 1:400
15 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT 30 min, RT -- continued
56
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Multiple Staining in Molecular Morphology 2.6.10 Suggested Immunoreagent List (continued) Reagent
Vendor and Code
Dilution Time/Temp.
Goat anti-rabbit Ig/GAL Goat anti-rabbit Ig/HRP Goat anti-rabbit, EnVision+/HRP Normal Goat Serum Normal Mouse Serum Normal Swine Serum Rabbit anti-biotin/HRP Rabbit anti-biotin/AP Rabbit anti-FITC Rabbit anti-FITC/HRP Rabbit anti-FITC/AP Rabbit anti-PE Serum-free protein blocking Sheep anti-DIG/HRP Sheep anti-DIG/AP Streptavidin/HRP Streptavidin/AP Streptavidin-Biotin Complex/HRP Streptavidin/GAL Swine anti-goat Ig/AP Swine anti-goat Ig/HRP Swine anti-rabbit Ig/AP Swine anti-rabbit Ig/HRP TSA Kit
SBA 4010-06 DakoCyto P0448 DakoCyto K4002/4003 DakoCyto X0907 DakoCyto X0910 DakoCyto X0901 DakoCyto P5106 DakoCyto D5107 DakoCyto V0403 DakoCyto P5100 DakoCyto D5101 BioGenesis 7374-2304 DakoCyto X0909 Roche 1 207 733 Roche 1 093 274 DakoCyto P0397 DakoCyto D0396 DakoCyto K0377 Roche 1 112 481 BioSource ACI 3405 BioSource ACI 3404 DakoCyto D0306 DakoCyto P0399 Perkin-Elmer NEL-700
1:10 30 min, RT 1:50 30 min, RT undiluted 30 min, RT 1:10 15 min, RT 1:10 15 min, RT 1:10 15 min, RT 1:50 30 min, RT 1:20 30 min, RT 1:1000 15 min, RT 1:50 30 min, RT 1:20 30 min, RT 1:200 15 min, RT Undiluted 15 min, RT 1:400 30 min, RT 1:100 30 min, RT 1:400 30 min, RT 1:100 30 min, RT 1:100 30 min, RT 1:40 30 min, RT 1:20 30 min, RT 1:50 30 min, RT 1:20 30 min, RT 1:50 30 min, RT
Note: DakoCyto = Dakocytomation; SBA = Southern Biotechnology Associates.
hydrogen peroxide 30% just before use. Stain for 5 to 20 min at room temperature. Result: brick-red reaction product, soluble in alcohol and xylene. Comment: the used concentration of AEC chromogen is may be higher than most laboratories usually apply; this higher concentration of chromogen will result in a more intensely stained reaction product, without causing nonspecific background problems. 2.6.11.2 Protocol for HRP/DAB, after Graham and Karnovski [55] Dissolve 5 mg 3,3′-diaminobenzidine (DAB) (Sigma D5637) in 10 ml Tris-HCl buffer (50 mM, pH 7.8); add 10 µl hydrogen peroxide 30% just before use. Stain for 5 to 20 minutes at room temperature. Result: brown-yellow reaction product,
aqueously and organically insoluble. Notes: color shifting and intensification using metal ions is described by Hsu and Soban [56] (blue-black) and Nemes [57] (green). 2.6.11.3 Protocol for HRP/TMB, modified from Buckel and Zehelein [58] Dissolve 24 mg 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma T2885) and 80 mg dioctyl sodium sulphosuccinate (Sigma D0885) in 10 ml warm ethanol (∼ 60°C). Mix with 30 ml citrate/phosphate buffer (100 mM, pH 5). Add 10 µl hydrogen peroxide 30% just before use. Stain for 2 to 10 min at room temperature. Result: bluegreen reaction product, aqueously soluble. Note: after staining, wash briefly in distilled water and dry section completely. Soak in xylene and mount up organically. 57
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Molecular Morphology in Human Tissues: Techniques and Applications 2.6.11.4 Protocol for AP/Fast Blue/Fast Red, modified from Burnstone [59]; Boorsma [9]) Dissolve 20 mg naphthol-ASMX-phosphate (Sigma N4875) in N,N-dimethylformamide and mix with Tris-HCl buffer (100 mM, pH 8.5); store 5- ml aliquots at –20°C. Add to one 5-ml aliquot just before use: 1 mg Fast Blue BB (Sigma F3378) or 5 mg Fast Red TR (Sigma F1500); 0.5 mM levamisole (Sigma L9756) should be included for cryostat sections (2.5 µl of 1 M concentrate for 5 ml) to inhibit endogenous alkaline phosphatase activity [43]. Stain for 5 to 20 min at room temperature. Result: blue (Fast Blue BB) or red (Fast Red TR) reaction product, soluble in alcohol and xylene. The reaction product obtained with Fast Red is also highly fluorescent [51, 60]. Notes: levamisole may also slightly inhibit the marker alkaline phosphatase activity [45]; therefore, do not include levamisole for paraffin sections. When phosphate buffered saline is used throughout the incubation steps, the buffer salts should be washed out prior to the enzyme visualization using Tris-HCl buffer because phosphate ions have an inhibitory effect on alkaline phosphatase activity. 2.6.11.5 Protocol for AP/NBT/BCIP, modified from McGadey [61] Dissolve 18 mg 5-bromo-4-chloro-3indolyl-phosphate (BCIP) (Boehringer 760 994) in 1 ml N,N-dimethylformamide; store at −20°C. Prepare: nitro blue tetrazolium (NBT) (Sigma N6876) 5 mg/ml in aqua dest. (store at −20°C) and Tris-HCl buffer (100 mM, pH 9.5), including magnesium chloride (10 mg/ml). Mix 100 µl BCIP substrate solution with 9.34 ml TrisHCl/Mg buffer and 0.66 ml NBT solution. Stain for 5 to 20 min at room tem-
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perature. Result: purple-blue reaction product, partly soluble in alcohols and xylene. VectaMount (organic medium without alcohol or xylene) is very useful for mounting. See also notes concerning levamisole and phosphate ions in Fast Blue/Fast Red section. 2.6.11.6 Protocol for AP/New Fuchsin, modified from Feller et al. [62] Dissolve 35 mg naphthol-ASBI-phosphate (Sigma N2250) in 0.42 ml N,Ndimethylformamide and mix with TrisHCl buffer (50 mM, pH 9.7), including 378 mg/50 ml 2-amino-2-methyl-1,3propanediol (Sigma A9754) and 600 mg/50 ml sodium chloride; store 5-ml aliquots at –20°C. Mix 83 µl freshly prepared New Fuchsin (Merck 4041) (12.5 mg/2 ml 2 N HCl), with 52 µl sodium nitrite solution (Merck 6549) (20 mg/ml aqua dest.); let stand for 2 min for diazonium reaction. Mix with 5 ml buffer/substrate solution; check pH to be 8.7. Stain for 5 to 20 min at room temperature. Result: red reaction product, slightly soluble in alcohol (see: description of red/turquoise color combination). See also notes concerning levamisole and phosphate ions in the Fast Blue/Fast Red section. 2.6.11.7 Protocol for β-Galactosidase/ X-gal, after Bondi et al. [63] Dissolve 20 mg 5-bromo-4-chloro-3(X-gal) indolyl-β-D-galactopyranoside (Boehringer 651 745) in 1 ml N,Ndimethylformamide; store at –20°C. Prepare 50 ml phosphate buffered saline, pH 7.4, including 10 mg magnesium chloride·6H2O, 49.5 mg potassium ferrihexacyanide, and 63.5 mg potassium ferro-
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Multiple Staining in Molecular Morphology hexacyanide; store at +4°C. Mix 20 µl Xgal substrate with 1 ml ironphosphate solution. Stain for 15 to 60 min at 37°C. Result: turquoise reaction product, aqueously and organically insoluble. 2.6.11.8 Protocol for β-Galactosidase/ Bluo-Gal, after Aguzzi and Theuring [38] Dissolve 20 mg 5-bromo-β-D-galactoside (Bluo-Gal) (Sigma B4387) in 1 ml N,N-dimethylformamide; store at –20°C. Mix 10 µl X-gal substrate with 1 ml ironphosphate solution (see X-gal section). Stain for 15 to 60 min at 37°C. Result: blue reaction product, aqueously and organically insoluble. 2.6.12 Commercial Visualization Systems HRP – AEC: DakoCytomation AEC+ substrate chromogen K3461. HRP – DAB: DakoCytomation DAB+ liquid chromogen K3467. HRP – NovaRed: Vector Laboratories substrate kit SK-4800.
HRP – TMB: HistoMark True Blue peroxidase substrate, Kirkegaard & Perry Laboratories 71-00-64. AP – Fast Red: DakoCytomation Fast Red substrate system K/0597/K0699. See also DakoCytomation Permanent Red substrate system K0695 that can be mounted up organically. Both reaction products are also highly fluorescent [51]. AP – New Fuchsin: DakoCytomation New Fuchsin substrate system K0596. AP – NBT/BCIP: DakoCytomation NBT/BCIP substrate system K0598, or Roche 1442 074. AP – Vector Red: Vector Laboratories SK5100: Prepare 100 mM Tris-HCl buffer pH 8.2 (1.2 g Tris/100 ml, adjusted with 2 N HCl). Add 0.1% Tween 20 for sharper localization of reaction product. This reaction product is also highly fluorescent [51]. AP – Vector Blue: Vector Laboratories SK5300; see Vector Red for Tris-HCl buffer. β-Glactosidase/X-gal: HistoMark X-gal detection kit, Kirkegaard & Perry Laboratories 54-43-00.
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14. Vandesande, F., Immunohistochemical double staining techniques, in Immunohistochemistry, IBRO Handbook, Cuello, A.C., Ed., John Wiley & Sons, Chichester, U.K., 1983, 257. 15. Sternberger, L.A. and Joseph, S.A., The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal, J. Histochem. Cytochem., 27, 1424, 1979. 16. Valnes, K. and Brandtzaeg, P., Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels, Histochem. J., 16, 477, 1984. 17. Krenács, T. et al., Double and triple immunocytochemical labelling at the light microscopical level in histopathology, Histochem. J., 22, 530, 1990. 18. Mullink, H. et al., Double immunoenzyme staining methods with special reference to monoclonal antibodies, in Application of Monoclonal Antibodies in Tumor Pathology, Ruiter, D.J., Fleuren, F.J., and Warnaar, S.O., Eds., Martinus Nijhoff Publishers, Dordrecht, the Netherlands, 1987, 37. 19. Pastore, J.N. et al., A rapid immunoenzymne double labeling technique using EPOS reagents, J. Histotechnol., 27, 1424, 1995. 20. Lan, H.Y. et al., A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody cross-reactivity and retrieve antigens, J. Histochem. Cytochem., 43, 97, 1995. 21. Heras, A., Roach, C.M., and Key, M.E., Enhanced polymer detection system for immunohistochemistry (abstract), Lab. Invest., 72, 165A, 1995. 22. Newman, G.R., Jasani, B., and Williams, E.D., Multiple hormone storage by cells of the human pituitary, J. Histochem. Cytochem., 37, 1183, 1989. 23. Falini, B. et al., Description of a sequential staining procedure for double immuno-enzymatic staining of pairs of antigens using monoclonal antibodies, J. Immunol. Meth., 93, 265, 1986. 24. Van der Loos, C.M., Das, P.K., and Houthoff, H.-J., An immunoenzyme triple staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect and avidin-biotin complex (ABC) technique, J. Histochem. Cytochem., 35, 1199, 1987. 25. Van der Loos, C.M. et al., Multiple immunoenzyme staining techniques. Using of fluoresceinated, biotinylated and unlabeled monoclonal antibodies, J. Immunol. Meth., 117, 45, 1989.
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Multiple Staining in Molecular Morphology 26. Campbell, G.T. and Bhatnagar, A.S., Simultaneous visualization by light microscopy of two pituitary hormones in a single tissue section using a combination of indirect immunohistochemical methods, J. Histochem. Cytochem., 24, 448, 1976. 27. Mason, D.Y. and Sammons, R., Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents, J. Clin. Pathol., 31, 454, 1978. 28. Tidman, N. et al., Delineation of human thymocyte differentiation pathways utilizing doublestaining techniques with monoclonal antibodies, Clin. Exp. Immunol., 45, 457, 1981. 29. Van der Loos, C.M. et al., Use of commercially available monoclonal antibodies for immunoenzyme double staining, Histochem. J., 20, 409, 1988. 30. Van der Loos, C.M., Naruko, T., and Becker, A.E., The use of enhanced polymer one-step staining staining (EPOS) reagents for immunoenzyme double-labelling, Histochem. J., 28, 709, 1996. 31. Gillitzer. R., Berger, R., and Moll, H., A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling, J. Histochem. Cytochem., 38, 307, 1990. 32. Van der Loos, C.M. and Göbel, H., Application of DAKO Animal Research Kit (ARK) for biotinylation of mouse primary antibodies, to be used in a multistep double staining method for human tissue specimens, J. Histochem. Cytochem., 48, 1431, 2000. 33. Farmilo, A.J. and Stead, R.H., Fixation in immunocytochemistry, in Immunochemical Staining Methods, DAKO Handbook, Naish, S.J., Ed., 1989, 24. 34. Shi, S.-R., Cote, R.J., and Taylor, C.R., Antigen retrieval: current perspectives, J. Histochem. Cytochem., 49, 931, 2001. 35. Van den Brink, G.R. et al., Helicobacter pylori co-localizes with MUC5AC in the human stomach, Gut, 46, 601, 2000. 36. Scopsi, L. and Larsson, L.-I., Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods, Histochemistry, 84, 221, 1986. 37. Malik, N.J. and Daymon, M.E., Improved double immunoenzyme labelling using alkaline phosphatase and horseradish peroxidase, J. Clin. Pathol., 35, 1092, 1982.
38. Aguzzi, A. and Theuring, F., Improved in situ beta-GALactosidase staining for histological analysis of transgenic mice, Histochemistry, 102, 477, 1994. 39. Lehr, H.-A. et al., Differential chromogen display and analysis in double immunohistochemical stains using Adobe Photoshop, J. Histochem. Cytochem., 47, 119, 1999. 40. Lehr, H.-A. et al., Application of Photoshopbased image analysis to quantification of hormone receptor expression in breast cancer, J. Histochem. Cytochem., 45, 1, 1997. 41. Ornberg, R.L., Woerner, B.M., and Edwards, D.A., Analysis of stained objects in histopathological sections by spectral imaging and differential absorption, J. Histochem. Cytochem., 47, 1307, 1999. 42. Levenson, R., Cronin, P.J., and Pankratov, K.K., Spectral imaging for brightfield microscopy, Proc. SPIE, 4959 (in press). 43. Wolber, R.A. and Lloyd, R.V., Cytomegalovirus detection by non-isotypic DNA in situ hybridization and viral antigen immunostaining using a two-color technique, Hum. Pathol., 19, 736, 1988. 44. Borgers, M., The cytochemical application of new potent inhibitors of alkaline phosphatase, J. Histochem. Cytochem., 21, 812, 1973. 45. Thisted, M., The use of levamizole in immunohistochemistry, Clin. Lab. Int., 19, 37, 1995. 46. Li, C.-Y., Ziesmer, S.C., and Lazcano-Villareal, O., Use of azide and hydrogen peroxide as inhibitor for endogenous peroxidase in the immunoperoxidase method, J. Histochem. Cytochem., 35, 1457, 1987. 47. Andrew, S.M. and Jasani, B., An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers. Application to immunoperoxidase studies on eosinophil-rich tissue preparations, Histochem. J., 19, 426, 1987. 48. Wood, G.S. and Warnke, R., Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems, J. Histochem. Cytochem., 29, 1196, 1981. 49. Hsu, S.-M., Raine, L., and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29, 577, 1981.
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Molecular Morphology in Human Tissues: Techniques and Applications 50. Cattoretti, G., Pileri, S., Parravicini, C., Becker, M.H.G., Poggi, S., Bifulco, C., Key, G., D’Amato, L., Sabattini, E., Feudale, E., Reynolds, F., Gerdes, J., and Rilke, F., Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections, J. Histopathol., 171, 83, 1993. 51. Van der Loos, C.M. and Becker, A.E., Double epi-illumination microscopy with separate visualization of two antigens: a combination of epipolarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining, J. Histochem. Cytochem., 42, 289, 1994. 52. Streefkerk, J., Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol, J. Histochem. Cytochem., 20, 829, 1972. 53. Sander, S., Andersson, J., and Andersson, U., Assessment of cytokines by immunofluorescence and paraformaldehyde-saponin procedure, Immunol. Rev., 119, 65, 1991. 54. Graham, R.C., Lundholm, U., and Karnovsky, M.J., Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole, J. Histochem. Cytochem., 13, 150, 1965. 55. Graham, R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubes of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14, 291, 1966. 56. Hsu, S.-M. and Soban, E., Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry, J. Histochem. Cytochem., 30, 1079, 1982. 57. Nemes, Z., Intensification of 3,3′-diamino-benzidine precipitation using the ferric ferricyanide reaction, and its application in the doubleimmunoperoxidase technique, Histochemistry, 86, 415, 1987. 58. Buckel, P. and Zehelein, E., Expression of pseudonomas fluorescens D-galactose dehydrogenase in E. coli, Gene, 16, 149, 1981. 59. Burnstone, M.S., Histochemical demonstration of phosphatases in frozen sections with naphthol AS-phosphatases, J. Histochem. Cytochem., 9, 146, 1961. 60. Speel, E.-J., Schutte, B., Wiegant, J., Raemakers, F.C.S., and Hopman, A.H.N., A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-Fast Red reaction, J. Histochem. Cytochem., 40, 1299, 1992. 61. McGadey, J., A tetrazolium method for non-specific alkaline phosphatase, Histochemistry, 23, 180, 1970.
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62. Feller, A.C., Parwaresch, M.R., Wacker, H.H., Radzun, H.-J., and Lennert, K., Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils, Histochem. J., 15, 557, 1983. 63. Bondi, A., Chieregatti, G., Eusebi, V., Fulcheri, E., and Bussolati, G., The use of beta-GALactosidase as a tracer in immunohistochemistry, Histochemistry, 76, 153, 1982.
ABBREVIATIONS Fluorochromes – Enzymes – Haptens – Complexes AP APAAP
alkaline phosphatase alkaline phoshatase anti-alkaline phosphatase complex DIG digoxigenin FITC fluoresceine isothiocyanate HRP horseradish peroxidase beta-GAL beta-GALactosidase PAP peroxidase anti-peroxidase complex Substrates – Chromogens – Buffers AEC BCIP
3-amino-9-ethylcarbazole 5-bromo-4-chloro-3-indolylphosphate Bluo-GAL 5-bromo-indolyl-galactoside DAB 3,3′-diaminobenzidine IGSS immunogold-silver staining NBT nitro blue tetrazolium PBS phosphate buffered saline TBS Tris-HCl buffered saline TMB 3,3′,5,5′-tetramethylbenzidin X-gal 5-bromo-4-chloro-3-indolylgalactopyranoside Antibodies Ig GAM GAR RAM
immunoglobulin goat anti-mouse immunoglobulin goat anti-rabbit immunoglobulin rabbit anti-mouse immunoglobulin SAG swine anti-goat immunoglobulin Strep streptavidin StrepABC streptavidin-biotin complex
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Multiple Staining in Molecular Morphology Reagent Sources Aurion BV, Wageningen, the Netherlands www.aurion.nl Becton Dickinson, San Jose, CA www.bdcis.com Biogenesis Poole, U.K. www.biogenesis.co.uk BioGenex Laboratories, San Ramon, CA www.biogenex.com BioSource Int., Camarillo, CA www.biosource.com Roche, Mannheim, Germany www.roche.com Chemicon-Cymbus, Temecula, CA www.chemicon.com DakoCytomation A/S, Glostrup, Denmark www.dakocytomation.co Carpinteria, CA
us.dakocytomation.com ImmunoVision, Daly City, CA www.immunovisiontech.com Kirkegaard & Perry Laboratories, Gaithersburg, MD www.kpl.com LabVision-NeoMarkers, Fremont, CA www.labvision.com Novocastra Labs. Ltd., Newcastle upon Tyne, U.K. www.novacastra.co.uk Southern Biotechnology Associates, Birmingham, AL www.southernbiotech.com Vector Laboratories, Burlingame, CA www.vectorlabs.com
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Enzyme-Based Fluorescence Amplification for Immunohistochemistry and In Situ Hybridization Kevin A. Roth and Denis G. Baskin
3.1 INTRODUCTION Immunohistochemical (IHC) and in situ hybridization (ISH) detection in human tissue sections have traditionally been performed with chromogenic substrates due to significant autofluorescence in many human tissues and the relatively poor sensitivity and photostability of conventional fluorescence detection procedures. Recent advances in enzyme-based amplification methods, novel fluorophores, and improved detection protocols have dramatically increased the utility of fluorescence localization procedures in human tissue sections. This chapter provides detailed protocols for enzyme-enhanced immunohistochemical and ISH detection and discusses practical solutions to common problems encountered using these sensitive methods. Detection of specific antigens and mRNA sequences in human tissue sections typically requires the use of sensitive IHC and ISH techniques. Radiolabeled probes or antibodies provide exquisite sensitivity 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
and have proven particularly useful in the detection and localization of low abundance mRNA expression in tissue sections [1]. Protocols utilizing radioactive isotopes, however, are costly, technically demanding, and ultimately produce signals that have relatively poor spatial resolution and low signal-to-noise ratios. In contrast, fluorophore-labeled probes and antibodies are more convenient to use and provide exquisite spatial localization of signal but poor sensitivity restricts their application to relatively abundant mRNAs and antigens. To overcome the limitations of radioactive detection methods and the poor sensitivity of fluorescently labeled probes and antibodies, many ISH and IHC protocols utilize enzyme-catalyzed conversion of soluble substrates into insoluble, colored reaction products to localize specific mRNA and protein expression [2]. In these protocols, enzyme-linked primary (probe or antibody) or secondary detection reagents (such as streptavidin or secondary antibody) are used to precipitate chromogen
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Molecular Morphology in Human Tissues: Techniques and Applications near sites of target expression. The most commonly used enzymes for ISH and IHC detection are horseradish peroxidase (HRP) and alkaline phosphatase (AP) and a wide variety of HRP and AP substrates, producing a range of colored reaction products have been effectively used to localize specific mRNA and antigen expression in human tissue sections. Although fairly sensitive and easy to perform chromogenic ISH and IHC protocols have been described, enzyme-enhanced fluorescence detection methods offer several potential advantages over these techniques. The presence of pigment in various cell types, for example, neuromelanin in substantia nigra neurons, may interfere with the transmitted light detection of chromogen [3]. Multi-label detection methods utilizing different colored chromogens are compromised by potential masking of target reactivity by previously deposited chromogen and difficulty discerning two, or more, chromogens within individual cells when target co-localization occurs [4]. Finally, on a per-molecule basis, detection of fluorescent light emission from a fluorophore may be orders of magnitude more sensitive than detection of chromogen light absorbance. In enzyme-linked immunoadsorbent assays (ELISA), the use of non-fluorescent enzyme substrates that are converted into fluorescent reaction products has been reported to produce over a 100-fold increase in sensitivity over that achieved with colorimetric substrates [5, 6]. This “inherent” sensitivity of fluorometric methods is not readily appreciated in conventional ISH and IHC techniques because the number of fluorescent molecules directly linked to the primary or secondary detection reagent(s) is limited compared to the amount of chromogen deposited by enzymatic amplification. An 66
ideal situation occurs, however, when enzyme amplification is coupled, either directly or indirectly, with fluorescence detection. A major limiting factor to enzymeenhanced fluorescence ISH and IHC procedures has been the fact that most HRP and AP substrates do not form fluorescent products. Furthermore, the few fluorescent end-products that have been described are typically soluble and diffuse rapidly from sites of enzymatic conversion, thus precluding their use as in situ localization reagents [6]. An approach that does not require an insoluble and fluorogenic enzyme reaction is to “couple” or “capture” the AP or HRP reaction product with additional reagents that impart these properties [6, 7]. Papadimitriou et al. reported that the soluble fluorescent HRP reaction product of non-fluorescent homovanillic acid could be precipitated as a complex salt in the presence of lead ions and Rhodamine G or Rhodamine B and produced a fluorescent signal that was useful for localizing peroxidase activity in tissue sections [8]. This procedure is complicated by the fact that it is relatively insensitive, has a low signal-to-noise ratio, is difficult to perform, and the precipitated product is fluorochromic — not fluorogenic alone [6]. Metal salts have also been used in combination with fluorogenic AP substrates to generate localizable fluorescent signals but were found to have limited applicability due to poor sensitivity [6]. More useful capture reagents for generating localizable AP fluorescent signals have been developed [9, 10]. In one such procedure, naphthol ASMX phosphate substrate is used in combination with the diazonium salt Fast Red TR to produce a highly fluorescent precipitate with significant photostability that has been used for both IHC and ISH detection [11].
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Enzyme-Based Fluorescence Amplification Several problems with enzyme-amplified fluorescence procedures that require coupling reagents have limited the adoption of these approaches as widespread tools for ISH and IHC. These difficulties include a significant diffusion of reaction product away from the sites of enzymatic activity, as well as the production of large fluorescent precipitates that limit subcellular localization of target, and, in many protocols, high non-specific background staining. For these reasons, and the relative complexity of coupling procedures, the direct deposition of enzyme reaction product has been the preferred method. Nevertheless, a few examples of AP and HRP substrates forming insoluble fluorescent reaction products have proven useful for histochemistry. Burstone [7] reported more than 40 years ago the use of 5,6,7,8β-tetralol carboxylic acid-β-naphthylamide phosphate to localize endogenous AP activity in tissue sections by fluorescence microscopy, but this compound has not been widely used. Larsson and Hougaard [12] successfully used fluorescent AP reaction products for a double IHC and ISH protocol to detect peptides and their mRNAs in gut endocrine cells. We recently found that soluble p-hydroxyphenylcontaining organic compounds react with HRP to form insoluble fluorescent reaction products [13]. Interestingly, the deposited reaction product initially shows limited fluorescence that is significantly enhanced by near-ultraviolet illumination. Once “activated,” the deposited fluorescent signal is remarkably photostable and shows an apparent large Stokes shift. The major limitation of p-hydroxyphenyl-containing HRP substrates is their relatively low sensitivity, in particular compared with that of tyramide signal amplification (TSA) detection (see below).
Recently, Haugland and colleagues [14–16] described a sensitive depositable fluorogenic reagent for detecting AP activity in both IHC and ISH applications. This compound, 2-(5′-chloro-2-phosphoryloxyphenyl)-6-chloro-4 (3H)-quinazolinone, or ELF (Enzyme-Labeled-Fluorescence)-97 phosphate has low intrinsic fluorescent properties but is converted to a highly fluorescent compound, ELF-97 alcohol. The ELF-97 reaction product is a fluorescent precipitate with a large Stokes shift and significantly more photostability than fluorescein. It also exhibits considerably less diffusion than that observed with AP substrates requiring a capture reagent. When viewed with appropriate fluorescence filters, the ELF-97 reaction product appears as a very bright green fluorescence with excitation and emission maxima that are widely separated from those of Cy3, with no “bleed-through” of signals. Therefore, ELF-97 can be used as a green fluorochrome to complement Cy3 in double labeling protocols [17]. The utility of enzyme-based fluorescence amplification procedures was dramatically increased with the development of tyramide-signal-amplification (TSA; Perkin-Elmer, http://las.perkinelmer.com/) methodologies. TSA was discovered by Bobrow and colleagues and is based on the ability of HRP to convert labeled tyramine-containing substrate to an oxidized, short-lived free radical that can covalently bind to tyrosine residues at or near the HRP [18–20]. Because tissue-bound tyrosine-tyramine heterodimer shows only limited fluorescence, maximal sensitivity is achieved with fluorophore-labeled tyramine. Numerous studies have reported the successful application of TSA techniques to ISH and IHC detection [21–26]. TSA is fundamentally different from other enzyme-enhanced fluorescence detection procedures in that the fluorescent reaction 67
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Molecular Morphology in Human Tissues: Techniques and Applications product is covalently bound to the solidphase tissue section. Also, because a critical concentration of reaction product is not required to form an insoluble precipitate, lower detection limits can be achieved. An alternative to the direct localization of fluorescently labeled tyramide is the use of hapten-labeled tyramide and fluorescently labeled secondary detection reagents. This technique offers significant experimental flexibility and permits coupling of TSA to additional enzyme amplification methods [27]. Regardless of the label, binding of oxidized tyramide with tyrosine residues in the solid phase (i.e., on the tissue section) is in competition with tyramide dimerization in solution. Gross and Sizer [28] demonstrated that tyramine, tyrosine, and other substrates with free phenolic groups were oxidized by HRP to form dimers. This dimerization reaction forms the basis of very sensitive solution-phase fluorescence measurements of HRP activity and H2O2 concentration because these non-fluorescent substrates are converted into highly fluorescent dimers [29, 30]. In IHC and ISH applications, tyramide dimer formation produces non-specific background signals and decreases specific tyramide binding. To minimize tyramide dimer formation, an appropriately low substrate concentration is required so that specific binding of the reactive tyramide intermediate to solid-phase tyrosines is promoted. Optimization of tyramide concentration is very important when performing IHC or ISH with commercially available TSA Plus reagents (Perkin-Elmer Life Sciences Products). TSA Plus contains an optimized HRP reaction buffer and enhancers that dramatically increase catalysis of labeled tyramide by HRP. These reagents are based on the discovery that inorganic salts and certain organic molecules, alone or in combination, produced a marked increase in tyramide deposition in TSA protocols [31]. 68
In many instances, these reagents are critical in achieving the necessary sensitivity for TSA ISH in human tissue sections. A great advantage of TSA ISH and IHC detection is experimental flexibility. TSA can be performed with any of a variety of fluorescently conjugated tyramides, or it can be combined with additional amplification techniques to further increase detection sensitivity. Yang et al. (in 1999) reported that the combination of TSA and alkaline phosphatase for FISH with digoxigenin-labeled riboprobes yielded strong hybridization signals for mRNAs that are expressed at very low abundance, thereby achieving the high sensitivity normally associated with radioactive probes but with the cellular resolution provided by chromogenic enzymatic detection [32]. We have combined HRP-based biotinylated tyramide deposition with streptavidin Quantum Dots for IHC [33] and with streptavidin AP-catalyzed ELF-97 detection for extremely sensitive ISH detection [27, 34, 35]. Protocols for TSA IHC, TSA ISH, ELF97 IHC, and dual TSA/ELF-97 ISH are presented in Section 3.2. Finally, although TSA has been the most popular enzyme-enhanced fluorescence detection procedure available, other HRP substrates have been used for fluorescencebased catalyzed analyte reporter deposition (CARD). We recently found that 4-(4hydroxystyryl) pyridine-containing substrates are very sensitive fluorescent HRP detection reagents and, due to their large Stokes shift and photostability, offer some advantages over fluorescently conjugated tyramide substrates [36]. These and other novel substrates are likely to expand the application of enzyme-enhanced fluorescence techniques to IHC and ISH detection in human tissue sections.
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747; Perkin-Elmer Life Sciences Products). Fluorescent Nuclear Counterstain: Bisbenzimide (Hoechst 33258; Code No. B-2883, Sigma, St. Louis, MO). Clearing Agent: CitriSolv (Code No. 22-143975; Fisher Scientific).
Protocol A: TSA IHC
3.2.1.1 Materials and Reagents Antigen retrieval buffer: 10 mM citrate buffer, pH 6.0: Stock Solution A: 0.1 M Citric Acid, monohydrate (Sigma C-7129; 2.1 g/100 ml). Store at 4°C. Stock Solution B: 0.1 M Sodium Citrate (Sigma S-4641; 14.75 g/500 ml). Store at 4°C. Working solution consists of 9 ml Solution A, 41 ml Solution B, and 450 ml distilled H2O. Phosphate Buffered Saline (PBS), pH 7.2: 10X PBS Stock: 80.0 g NaCl, 2.0 g KC1, 14.2 g Na2HPO4, 2.0 g KH2PO4, and 0.1 g NaN3 in 1 L of distilled H2O. Adjust pH to 7.2 with NaOH. Dilute to 1× PBS with distilled H20. PBS-Blocking Buffer: 1.0 g bovine serum albumin, 0.2 g nonfat powdered skim milk, 0.3 ml Triton X-100, in 100 ml 1X PBS. Additional Reagents: Hydrophobic slide marker: PAP Pen (Code No. 195504; Research Products International, Mount Prospect, IL). Primary Antibodies: Monoclonal or polyclonal (various sources). Secondary Antibodies: HRP conjugated Affinipure antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). TSA Reagents: Standard TSA Systems (Code No. NEL700-705; PerkinElmer Life Sciences Products, Boston, MA); TSA Plus Systems (NEL 741-
3.2.1.2
Staining Procedure
Deparaffinize sections (CitriSolv, 2 × 10 min). 2. Rehydrate (isopropanol 3 × 5 min; running tap water 5 min; distilled water 5 min). 3. Citrate antigen retrieval as appropriate (20 min in steamer followed by 20 min cool-down). 4. Destroy endogenous peroxidase activity by incubating sections in 3.0% H2O2 in PBS at room temperature for 5 min. 5. Rinse in tap water, distilled water, and PBS (5 min each). 6. Incubate sections in PBS-BB for 30 min at room temperature to inhibit non-specific antibody binding. 7. Rapidly blot excess moisture around the tissue section with a paper towel and encircle the tissue with a PAP pen. 8. Immediately add an adequate volume of primary antibody diluted in PBSBB to the tissue section (20 to 200 µl, depending on tissue size). 9. Place sections in a humidified chamber for 12 to 24 hr at 4°C or for 60 to 90 min at room temperature. 10. Wash in PBS 3 × 5 min. 11. Apply HRP conjugated secondary antibody diluted in PBS-BB for 1 hr at room temperature (we resuspend the HRP labeled secondary antibody per the manufacturer’s recommendation and store it in aliquots at −70°C. 1.
69
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Molecular Morphology in Human Tissues: Techniques and Applications We typically use a 1:1000 to 1:2000 dilution of the stock for TSA IHC). 12. Wash in PBS 3 × 5 min. 13a. For standard direct TSA, the fluorophore-conjugated tyramide should be diluted in amplification buffer as recommended by the manufacturer. Apply the solution to the tissue for 5 to 10 min at room temperature. b. For TSA Plus direct TSA, the fluorophore-conjugated tyramide can either be diluted in Plus amplification buffer as recommended by the manufacturer and reacted on the tissue section for 5 to 10 min at room temperature, or it can be further diluted in Amplification buffer (thus lowering the tyramide concentration) and the reaction time can be extended to 30 to 60 min. For indirect TSA detection, either in the standard or Plus format, hapten-conjugated tyramide is deposited, followed by PBS washes and application of secondary detection reagents. Because of the multiple steps involved and the numerous variables that can potentially be modified, it is best to begin with the manufacturer’s recommended protocol before optimizing the individual steps for any specific application. 14. Wash in PBS 3 × 5 min (Nuclear counterstaining with Hoechst 33,258 may be performed by adding 1 µl of 2 mg/ml stock Hoechst 33,258 solution per 10 ml PBS during the second PBS wash). 15. Coverslip section in PBS/glycerol (1:1).
3.2.2
3.2.2.1 Materials and Reagents In addition to the materials and reagents described in Protocol A, the following are required: Trilogy deparaffinization, rehydration, and unmasking solution (Cell Marque, Hot Springs, AR; Code No. CMX833). Proteinase K (Invitrogen; Code No. 25530-015). HRP conjugated mouse anti-digoxin antibody (Jackson ImmunoResearch Laboratories; Code No. 200-032156). Mouse liver acetone powder (Sigma; Code No. L-8254). Hybridization buffer: 5X SSC, 50% formamide (Fisher; Code No. BP227500), 50 µg/ml salmon sperm DNA (Sigma; Code No. D7656), in DEPC treated water, pH 7.5 (pH adjusted with 0.1 N HC1). 3.2.2.2 1.
2.
3. 4. 5. 6.
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Protocol B: TSA ISH
Staining Procedure
Deparaffinize and antigen retrieve sections in Trilogy solution for 15 min in steamer, followed by an additional 15 min in fresh Trilogy solution. Wash slides twice in DEPC treated PBS for 5 min each (all PBS steps through probe hybridization should be performed with DEPC-treated PBS). Incubate slides with Proteinase K (10 µg/ml) in PBS for 15 min at room temperature. Wash slides in PBS for 2 × 5 min. Incubate slides in 80% acetone, 20% DEPC water for 15 min at −20°C. Wash sections in PBS for 5 min.
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Enzyme-Based Fluorescence Amplification 7. 8. 9.
10. 11. 12. 13. 14.
15.
16. 17. 18. 19. 20. 21. 22. 23.
Fix sections in 4% paraformaldehyde in PBS for 15 min. Wash in PBS for 5 min. Incubate sections in “active” 0.3% DEPC in PBS for 15 min (add 600 ml DEPC to 200 ml PBS 20 to 30 min prior to use). Wash in PBS for 5 min. Incubate in 0.5% Triton X-100 in PBS for 15 min. Wash in PBS for 5 min. Wash for 15 min each in 2X SSC and 5X SSC. Dilute digoxigenin-labeled riboprobe (typically to a final concentration of 100 to 500 ng/ml) in hybridization buffer and heat at 80°C for 5 min. Add 40 µl (a volume sufficient to cover the tissue) of diluted probe to section and coverslip (coverslips are pretreated for at least 2 hr in active 0.3% DEPC in H2O, rinsed in inactive DEPC-treated H2O, and airdried just prior to use). Hybridize sections 12 to 24 hr at 65°C in a humidified chamber containing 5X SSC. Place slides in 2X SSC preheated to 65°C and incubate at room temperature for 15 min. Wash in 1X SSC for 15 min at room temperature. Wash in PBS for 5 min (posthybridization PBS washes do not require DEPC treated PBS). Destroy endogenous peroxidase activity by incubating slides in 0.3% H2O2 in PBS for 15 min. Wash in PBS twice for 5 min each. Incubate sections in PBS-BB for 30 min. Add 100 to 200 µl (a volume sufficient to cover the tissue) of HRP-conjugated mouse anti-digoxin antibody diluted in PBS-BB and incubate either overnight at 4°C or at room tempera-
24. 25.
26. 27. 3.2.3
ture for 1 hr in a humidified chamber. (To minimize non-specific antibody binding, 30 min prior to use, add 10 mg mouse liver acetone powder per milliliter of diluted [1:2000] mouse anti-digoxin antibody. The liver acetone powder is pelleted with a tabletop centrifuge and the supernatant is added to the sections.) Wash sections in PBS 3 × 5 min each. Apply fluorescently labeled TSA Plus reagent to the sections following the manufacturer’s instructions. (The dilution of the tyramide Plus reagent and the length of the reaction should be individually optimized to achieve a maximal signal-to-noise ratio.) Wash 3 × 5 min each in PBS (sections may be counterstained with fluorescent nuclear dyes if desirable). Coverslip in PBS/glycerol (1:1). Protocol C: ELF-97 IHC
3.2.3.1 Materials and Reagents: Both paraffin sections and cryostat sections can be used. We have only used tissues fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer) [17]. Times given for rinses and incubations are flexible and can be adjusted empirically. The staining protocol described below is a general guideline. Requirements for the buffer and stop solutions are critical, and the manufacturer’s instructions should be carefully followed. Details and recipes for preparing the various solutions used in this procedure can be obtained and downloaded as a pdf file at the Molecular Probes Web site (http://www.probes.com). Blocking buffer: 30 mM Tris, 150 mM NaCl, 1% BSA, 0.5% Triton X-100, pH 7.5.
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Molecular Morphology in Human Tissues: Techniques and Applications Wash buffer: 30 mM Tris, 150 mM NaCl, 1% BSA, 0.05% Triton X100, pH 7.5. Pre-reaction wash buffer: 30 mM Tris, 150 mM NaCl, pH 7.5. Stop buffer: 25 mM EDTA, 1.0 mM levamisole, 0.05% Triton X-100 in PBS, pH 7.2. ELF 97 Immunohistochemistry Kit (E6600, Molecular Probes, Eugene, OR): contains all reagents needed for immunostaining. 3.2.3.2 1.
Staining Procedure
Deparaffinize sections and bring to water (or use cryostat sections). 2. Wash buffer, 30 min, at room temperature (3 × 10 min). 3. Blocking buffer, 30 min, room temperature. 4. Primary antibody (monoclonal or polyclonal) at appropriate dilution (determine empirically) diluted in wash buffer, overnight in refrigerator (use covered humid tray). 5. Wash buffer, 30 min, room temperature (3 × 10 min). 6. Biotinylated second antibody (not provided by the kit) diluted (10 µg/ml) in wash buffer, 30 min, room temperature. 7. Wash buffer, 30 min, room temperature (3 × 10 min). 8. Streptavidin solution (from kit) diluted 1:250 in wash buffer, prepared fresh, 30 min, room temperature. 9. Wash buffer, 30 min, room temperature (3 × 10 min). 10. Biotin-XX conjugate of AP from kit, 1:250 in wash buffer, 30 min, room temperature. 11. Wash with pre-reaction buffer (3 × 10 min). 12. Prepare ELF 97 phosphatase substrate (from kit) diluted 20X in the 72
Immunohistochemistry Reaction Buffer (supplied with the kit), as described by the manufacturer. Filter with ELF spin filters (or other 0.2micron spin filter). 13. Incubate sections in filtered substrate solution for 10 sec to 10 min. 14. Immediately stop the enzyme reaction by rinsing all slides in the stop buffer. This should be done by plunging slides into the buffer and repeating the rinse exhaustively (15 to 20 times over a 10- to 15-min period). 15. Blot remaining stop buffer and mount in the mounting medium supplied by the manufacturer. 3.2.4 Protocol D: Double IHC with ELF 97 When the ELF 97 procedure is combined with other immunofluorescence detection systems for multiple labeling, the ELF 97 step is always carried out as the second (or last) step. We have successfully used the following protocol to immunostain insulin and glucagon cells in the pancreatic islets of Langerhans [17] (Figure 3.1 [top left, right]). The method is conceptually and technically simple. 1.
2.
3.
Immunostain with primary antibodies to the first antigen, using antimouse IgG-Cy3 second antibodies for the fluorochrome, and any standard immunofluorescent protocol. Follow with the ELF-97 IHC protocol described above for the antibodies to the second antigen and mount in the mounting media supplied with the kit. View in a microscope equipped with filters for Cy3 and ELF 97 alcohol fluorescence.
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Figure 3.1 (Color Figure 3.1 follows page 106.) Immunocytochemistry and in situ hybridization with ELF method. Top panels: immunocytochemical double labeling of a single rat pancreatic islet of Langerhans for insulin (left) and glucagon (right), using a combination of Cy3 and ELF-97 fluorchromes. The insulin immunocytochemical staining (left) is revealed with Cy3 fluorescence and the glucagon labeling with ELF (right). Note that the excitation signal produced by the fluorescence of the ELF-97 reaction product does not overlap with that produced by Cy3. Bottom panel: in situ hybridization for leptin receptor in the rat brainstem using the ELF method and TSA amplification. Image shows large motor neurons of the hypoglossal nucleus labeled with in situ hybridization reaction product produced by the ELF-TSA procedure (yellow) for leptin receptor mRNA. The central canal is seen in top center of figure. Nuclei of cells are revealed by Hoechst 33258 dye.
3.2.5
Protocol E: TSA-ELF-97 ISH
ELF 97 can be used for ISH on conventional tissue sections with excellent results. Detailed protocols and reagents can be downloaded from the Molecular Probes website. Here we present a protocol for signal amplification by combining the ELF 97 ISH protocol with TSA amplification. The protocol that we use is presented here in brief form, as the procedure primarily involves using several standard protocols in tandem [34, 35] (Figure 3.1, bottom). For the ELF 97 ISH, we follow the protocol supplied by the manufacturer (details on the Molecular Probes website), so these do not need to be elaborated here. Refer to the Molecular Probes website for recipes for the
various buffers used for ELF 97 ISH. For abundant mRNAs, the TSA step can be omitted. The basic procedure is as follows: 1. 2. 3. 4.
First obtain sections of tissue that have been prepped for ISH. Prepare riboprobes containing biotinUTP. Perform a standard riboprobe ISH procedure. Amplify the biotin moiety using a TSA Biotin System kit (Perkin-Elmer Life Sciences Products, Boston, MA), following the manufacturer’s protocol (dilute streptavidin-HRP 1:100 with TNT buffer and apply to slides for 30 min at room temperature in a moist chamber). 73
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Molecular Morphology in Human Tissues: Techniques and Applications 5.
Wash slides 3 times for 5 min each in TNT buffer at room temperature. 6. Apply biotinyl tyramide 1:50 in the buffer supplied in the kit, 10 min, at room temperature. 7. Further amplify the biotin deposited as a result of the TSA protocol using ELF-97 mRNA in situ Hybridization Kit #2 (Molecular Probes). Start by placing slides in the blocking buffer recommended by the manufacturer (i.e., 30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% BSA, 0.5% Triton X100, 1 mM levamisole), 30 min in a moist chamber at room temperature. 8. Wash 3 times for 5 min each in the pre-reaction buffer containing 30 mM Tris-HCl (pH 7.4) with 150 mM NaCl, to remove residual BSA and detergent, both of which inhibit the ELF reaction. 9. Dilute ELF 97 substrate (from kit) 1:20 in the reaction buffer supplied with the kit and filter with a 20-µm spin filter. Combine additives A and B from the kit at a dilution of 1:500 and apply final substrate solution to sections for exactly 10 min in a moist chamber at room temperature. 10. Immediately stop the reaction by rinsing in stop buffer containing 100 mM Tris-HCl (pH 7.4), 25 mM EDTA, 0.5% Triton X-100, and 1% levamisole. 11. Wash extensively in stop buffer for 15 to 30 min, with several changes. 12. Blot excess liquid from slides and mount in the medium supplied with the kit. 3.2.6 Protocol F: Double ISH with TSA-ELF-97 The TSA-ELF 97 ISH protocol can be combined with a conventional digoxigenin FISH to visualize two mRNA species 74
simultaneously [34]. The protocol is straightforward and intuitive, and should pose no major difficulty if one first starts with single ISH protocols that work successfully as single FISH procedures. The same reagents are used as described above. To perform double ISH with the TSA-ELF 97 methods, carry out the following steps: 1.
Hybridize first with a cocktail containing (a) a digoxigenin-labeled riboprobe against one mRNA, and (b) a biotin-labeled riboprobe against a second mRNA. The relative ISH concentrations and conditions required for the respective riboprobes should be empirically determined from single ISH experiments. 2. Post hybridization wash 3 × 5 min each in SSC at room temperature. 3. TNT buffer with 0.1 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.05% Triton X-100. 4. Apply primary mouse anti-digoxigenin monoclonal IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:5000 in TNT buffer with 1% normal goat serum for 3 hr at 37°C. 5. Wash 3 × 5 min each in SSC at room temperature. 6. Apply goat anti-mouse IgG-Cy3 (Jackson ImmunoResearch), 1:200 in TNT buffer for 1 hr at 37°C. 7. Wash 3 × 5 min each in SSC at room temperature. 8. Check at microscope to verify labeling. 9. Perform TSA-ELF 97 protocol described above. 10. Dip in 1 µg/ml aqueous Hoechst 33258 (benzamide) for visualization of cell nuclei. 11. Mount in medium supplied in ELF 97 ISH kit.
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3.3 RESULTS TSA IHC and ISH protocols have effectively been used to localize specific antigens and mRNAs in human tissue sections as well as in sections from experimental animals. Our work has primarily involved neuronal tissues, and Figure 3.2 illustrates the TSA localization of neurons and astrocytes with cell-specific antibodies in human brain sections. Unlike enzyme-catalyzed deposition of chromogen that often produces a diffuse or smudgy signal due to diffusion of reaction product prior to precipitation, fluorescent tyramide deposition is typically crisp and granular. This may also reflect the fact that the low concentrations of antibody needed to detect antigen in TSA IHC, unlike in conventional fluorescent IHC procedures, are not antigen saturating and, thus, localization of antibody binding is spatially discrete.
TSA ISH is technically more demanding to perform than TSA IHC and because of the significant signal amplification required to detect less abundant mRNAs, significant effort may be required to achieve adequate signal-to-noise ratios using this technique. We have successfully employed the TSA Plus ISH protocol described above to detect specific mRNAs in human brain sections and a wide variety of experimental animal tissues. Examples of mRNA ISH localization in human and experimental animal tissue sections are shown in Figure 3.3. In both TSA IHC and ISH, nonspecific background signals may result from several factors. If excess HRP conjugated secondary antibody is used, diffuse fluorescent speckling may occur due to nonspecific tissue binding; Figure 3.4A. If the fluorescent tyramide concentration is too high or the TSA reaction too long, fluorescent tyramide dimers may form, which we have found have an affinity for connective tissue components including collagen and elastin; Figure 3.4B. The presence of either of these artifacts requires re-optimization of the TSA reaction conditions to achieve the appropriate results.
Figure 3.2 TSA IHC in human brain sections. Neurons and astrocytes were detected in formalin-fixed human cortex using mouse anti-NeuN (A) and rabbit anti-glial fibrillary acidic protein (B) antibodies, respectively, and direct TSA detection with cyanine 3-tyramide. (Scale bars = 50 microns.)
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Figure 3.3 TSA ISH in human brain and experimental animal tissue sections. Numerous cells in a formalin-fixed, paraffin-embedded section of human brain glioblastoma multiforme, a highly aggressive brain tumor, show Bcl-X mRNA expression (A; scale bar = 100 microns). A section of Bouin’s fixed, paraffin-embedded mouse embryonic brain shows expression of histone H4 mRNA in proliferating cells in the ventricular zone (B; scale bar = 50 microns). In both examples, mRNA was detected with specific digoxigenin-labeled cRNA probes and TSA Plus cyanine 3-tyramide.
Figure 3.4 TSA associated background. Diffuse, non-specific background speckling may occur if the TSA reagent concentrations are not sufficiently optimized. In this example, sections incubated without primary antibody still exhibited granular fluorescent tyramide background due to non-optimized detection conditions (A). The generation of tyramide dimers during the TSA reaction may result in binding of the fluorescent dimers to the tissue section. We have observed selective tyramide dimer binding to a variety of connective tissue elements, including collagen and elastin in arterial blood vessels (B). (Scale bars = 50 microns.)
3.4 TECHNICAL HINTS AND DISCUSSION 3.4.1 Choice of TSA Detection Methods An advantage of TSA detection is the tremendous experimental flexibility offered 76
to the experienced investigator. A disadvantage of TSA detection is the tremendous experimental flexibility offered to the inexperienced investigator. Because this chapter deals specifically with fluorescence detection, we will not discuss options for chromogenic detection or the combined use of TSA and immunogold-silver detection
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Enzyme-Based Fluorescence Amplification techniques. We recommend that investigators choose a TSA detection system based on the sensitivity required in their particular application and one’s willingness to devote sufficient time and effort to optimize the TSA protocol for the application. In general, standard TSA detection reagents are sufficient for most IHC methods and TSA Plus reagents are required for ISH protocols. As in our combined TSA/ELF-97 ISH protocol, TSA detection can be combined with other enzyme amplification techniques or multiple rounds of TSA can be performed (e.g., biotin tyramide deposition may be followed by streptavidin HRP incubation and fluorescent tyramide deposition) to achieve a tremendous level of signal amplification. We have performed sequential fluorescein-tyramide deposition followed by HRP-conjugated anti-fluorescein and cyanine 3-tyramide deposition to extend the dynamic range of fluorescence ISH detection. Using this technique, cells with abundant mRNA expression will be labeled with both fluorescein and cyanine 3, while cells with less abundant mRNA expression will only label with cyanine 3. Of course, the more sensitive the detection system, the more critical the optimization of reagent concentrations, reaction times, nonspecific blocking steps, buffer washes, and in particular, the inclusion of controls, both positive and negative.
destroyed with H2O2 incubation (3.0% H2O2 in PBS for 5 min or 0.3% H2O2 in PBS for 30 min) or by heating (1 min in boiling water or incubation in antigen retrieval buffer in a steamer). It is essential to include control slides to assess the adequacy of this step so as to avoid false colocalization results.
3.4.2
3.4.4
Multi-Label TSA Detection
Although this chapter has focused on single label fluorescent detection, protocols for multi-label IHC, ISH, and combined IHC and ISH are available [25, 27, 37–40]. A critical step in these procedures is the destruction of HRP enzymatic activity from prior TSA deposition steps before addition of HRP conjugated reagents for subsequent TSA deposition reactions. HRP activity is fairly labile and can be
3.4.3 Combined TSA and Quantum Dot Detection The options for sensitive fluorescence detection have recently increased with the development and commercial availability of quantum dots. These nanocrystals (commercially available as Qdots from Quantum Dot Corporation; Hayward, CA) have the optical properties of high brightness, photostability, narrow emission spectra, and an apparent large Stokes shift [41]. We recently reported the combined application of TSA and quantum dots for IHC detection [33]. In this procedure, biotin tyramide deposition is followed by Qdot conjugated streptavidin incubation that ultimately provides both tremendous signal amplification (TSA effect) and fluorophore stability (Qdot effect). Methods for TSA/Qdot ISH are readily apparent from simple modifications of the TSA ISH protocol provided in this chapter. ELF Detection Protocols
Although the ELF-97 method is potentially a very sensitive technique, the method is particularly predisposed to the formation of spurious fluorescent crystals that obscure the FISH signal. The timing of the ELF-97 reaction appears to be critical. Because the rate of the enzyme reaction is rapid, sometimes on the order of seconds, allowing it to continue too long can result in formation of large amounts of 77
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Molecular Morphology in Human Tissues: Techniques and Applications spurious background crystals [14, 15]. These can migrate away from the original site of deposition and cause spontaneous crystal growth, further increasing the nonspecific background signal. We have observed that nonspecific formation of these crystals occurs very rapidly, and this artifact can be reduced by closely adhering to the length of the ELF-97 reaction time. We adjusted our IHC protocol with the antibodies we have used for an incubation period of 8 min [34]. However, for some protocols, the staining time can be 5 to 10 sec. We prefer to adjust the reaction conditions and antisera dilutions for longer incubation periods, as these are easier to control and more forgiving of slight variations in time. When using the ELF method for the first time, it is good practice to empirically determine the optimum ELF-97 reaction time that produces a signal with low background and then adhere rigorously to this procedure. We have had best success using the reagents in the manner suggested by the manufacturer, and using the mounting medium supplied with the kit. We have also found that the length of the washing steps is critical; many failures (production of excessive large background crystals) result from inadequate washing. It is also important to use the preincubation in the blocking buffer recommended by the manufacturer to obtain good results. The manufacturer claims that rinsing the tissue in buffer containing 1% BSA will remove the signal and allow the reaction to be rerun. We have found that this procedure does remove some crystals, but appears not to rescue completely a section that has excessive background crystal formation. In general, this “destaining” procedure is not recommended as an approach for reducing background.
78
Washing exhaustively at the end of the enzyme reaction is critical for reducing spurious background crystal formation and for keeping the specifically deposited crystals small (otherwise, stained cells can show fluorescent needle-like crystals rather than a fine fluorescent precipitate). Because alkaline phosphatase is retained in the tissue after the reaction is stopped, high-pH mounting media should be avoided to prevent the nonspecific buildup of the fluorescent crystals over time. Because the ELF technique also has the potential for detecting endogenous alkaline phosphatase activity [16, 42], an inhibitor of alkaline phosphatase activity, such as levamisole, should be used in the ELF blocking buffer and wash buffer. The manufacturer warns against exposing the ELF-97 reaction precipitate to organic solvents, which can cause it to dissolve. We have observed that the reaction product also dissolves in the presence of excess TNT buffer. Detection of ELF 97 reaction product requires special filters that are available commercially. Acknowledgments
We would like to thank John Breininger, Cecelia Latham, and Barbara Klocke for expert technical assistance; Angela Schmeckebier for assistance in preparing this chapter; and Dr. Mark Bobrow (PerkinElmer Life Science Products) for expert advice and insights on TSA. K.A.R was previously supported in part by a grant from Perkin-Elmer Life Science Products. K.A.R.’s laboratory is supported by grants from the National Institutes of Health, NS35107 and NS41962. D.G.B.’s research is supported by Merit Review and Career Scientist programs of the Medical Research Service of the Department of Veterans Affairs and by NIH DK-17047.
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Enzyme-Based Fluorescence Amplification References 1.
Baskin, D. G. et al., Leptin receptor long form splice variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus, J. Histochem. Cytochem., 47, 353, 1999. 2. Baldino, F., Jr. et al., Enzyme histochemical detection of neuronal mRNA, in In Situ Hybridization in Neurobiology — Advances in Methodology, Eberwine, J.H., Valentino, K.L., and Barchas, J.D., Eds., Oxford University Press, New York, 1989. 3. Schneider, J. A. et al., Improved detection of substantia nigra pathology in Alzheimer’s disease, J. Histochem. Cytochem., 50, 99, 2002. 4. Hermiston, M. L. et al., Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy, J. Histchem. Cytochem., 40, 1283, 1992. 5. Yolken, R.H. and Stopa, P.J., Enzyme-linked fluorescence assay: ultrasensitive solid-phase assay for detection of human rotavirus, J. Clin. Microbiol., 10, 317, 1979. 6. Raap, A.K., Localization properties of fluorescence cytochemical enzyme procedures, Histochemistry, 84, 317, 1986. 7. Burstone, M.S., Postcoupling, noncoupling, and fluorescence techniques for the demonstration of alkaline phosphatase, J. Natl. Cancer Inst., 24, 1199, 1960. 8. Papadimitriou, J.M. et al., A new method for the cytochemical demonstration of peroxidase for light, fluorescence and electron microscopy, J. Histochem. Cytochem., 24, 82, 1976. 9. Dolbeare, F. et al., Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells, J. Histochem. Cytochem., 28, 419, 1980. 10. Murdoch, A. et al., Alkaline phosphatse-fast red, a new fluorescent label. Application in double labeling for cell surface antigen and cell cycle analysis, J. Immunol. Meth., 132, 45, 1990. 11. Speel, E.J.M. et al., A novel fluorescence detection method for in situ hyridization, based on the alkaline phosphatase-fast red reaction, J. Histochem. Cytochem., 40, 1299, 1992. 12. Larsson, L.I. and Hougaard, D.M., Combined non-radioactive detection of peptide hormones and their mRNA’s in endocrine cells, Histochemistry, 96, 375, 1991.
13. Bobrow, M.N. and Roth, K.A., Method of Permanent Fluorescent Assay, 09/526,414 [US 6,518,036 B1], 2003, United States. 14. Larison, K.D. et al., Use of a new fluorogenic phosphatase substrate in immunohisto-chemical applications, J. Histochem. Cytochem., 43, 77, 1995. 15. Paragas, V.B. et al., The ELF-97 alkaline phosphatase substrate provides a bright, photostable, fluorescent signal amplification method for FISH, J. Histochem. Cytochem., 45, 345, 1997. 16. Cox, W.G. and Singer, V.L., A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity, J. Histochem. Cytochem., 47, 1443, 1999. 17. Baskin, D.G. et al., Immunocytochemical double staining with the ELF method, Proc. Microsc. Soc. Am., 822, 1995. 18. Bobrow, M.N. et al., Catalyzed reporter deposition, a novel method of signal amplification: application to membrane immunoassays, J. Immunol., 125, 279, 1989. 19. Bobrow, M.N. et al., Catalyzed reporter deposition, a novel method of signal amplification. II. Application to immunoassays, J. Immunol., 137, 103, 1991. 20. Bobrow, M.N. et al., The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats, J. Immunol. Meth., 150, 145, 1992. 21. Adams, J.C., Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains, J. Histochem. Cytochem., 40, 1457, 1992. 22. Van Gijlswijk, R.P.M. et al., Fluorochromelabeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization, J. Histochem. Cytochem., 45, 375, 1997. 23. Komminoth, P. and Werner, M., Target and signal amplification: approaches to increase the sensitivity of in situ hybridization, Histochem. Cell Biol., 108, 325, 1997. 24. Van de Corput, M.P.C. et al., Fluorescence in situ hybridization using horseradish peroxidaselabeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection, Histochem. Cell Biol., 110, 431, 1998. 25. Speel, E.J.M., Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors, Histochem. Cell Biol., 112, 89, 1999.
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Molecular Morphology in Human Tissues: Techniques and Applications 26. Nakajima, H. et al., In situ hybridization of ATtailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mRNA in formalinfixed and paraffin-embedded tissues, Am. J. Pathol., 162, 381, 2003. 27. Breininger, J.F. and Baskin, D.G., Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification, J. Histochem. Cytochem., 48, 1593, 2000. 28. Gross, A.J. and Sizer, I.W., The oxidation of tyramine, tyrosine, and related compounds by peroxidase, J. Biol. Chem., 234, 1611, 1959. 29. Guilbault, G.G. et al., New substrates for the fluorometric determination of oxidative enzymes, Anal. Chem., 40, 1256, 1968. 30. Zaitsu, K. and Ohkura, Y., New fluorogenic substrates for horseradish peroxidase: rapid and sensitive arrays for hydrogen peroxide and peroxidase, Anal. Biochem., 109, 109, 1980. 31. Bobrow, M.N., Adler, K.E., and Roth, K.A., Enhanced Catalyzed Reporter Disposition, 09/434,742[6,372,937 B1], 2002, United States. 32. Yang, H. et al., An optimized method for in situ hydridization with signal amplification that allows the detection of rare mRNAs, J. Histochem. Cytochem., 47, 431, 1999. 33. Ness, J.M. et al., Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection, J. Histochem. Cytochem., 51, 981, 2003. 34. Baskin, D.G. et al., SOCS-3 expression in leptinsensitive neurons of the rat hypothalamus in response to food intake, Regulatory Peptides, 92, 9, 2000.
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35. Grill, H.J. et al., Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake, Endocrinology, 143, 239, 2002. 36. Bobrow, M.N. and Roth, K.A., 4-(4-Hydroxystyryl) Pyridine-Containing Substrates for an Analyte Dependent Enzyme Activation System, 09/526,594[6,355,443 B1], 2002, United States. 37. Shindler, K.S. and Roth, K.A., Double immunofluorescent staining using two unconjugated primary antisera raised in the same species, J. Histochem. Cytochem., 44, 1331, 1996. 38. Zaidi, A.U. et al., Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification, J. Histochem. Cytochem., 48, 1369, 2000. 39. Roth, K.A., In situ detection of apoptotic neurons, in Neuromethods, Vol. 37: Apoptotis Techniques and Protocols, LeBlanc, A.C., Ed., Humana Press, Inc., Totowa, NJ. 40. Schad, A. et al., Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISHCARD) and immunohistochemistry (IHC)/ Immunofluorescence (IF), J. Histochem. Cytochem., 51, 751, 2003. 41. Watson, A. et al., Lighting up cells with quantum dots, BioTechniques, 34, 296, 2003. 42. Telford, W.G. et al., Detection of endogenous alkaline phosphatase activity by intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate, Cytometry, 37, 314, 1999.
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Gold Cluster Labels and Related Technologies in Molecular Morphology James F. Hainfeld and Richard D. Powell
4.1 INTRODUCTION: WHY CLUSTER LABELS? Although intensely colored, even the largest colloidal gold particles are not, on their own, sufficiently colored for routine use as a light microscopy stain; only with very abundant antigens or with specialized illumination methods can bound gold be seen [1]. Colloidal gold probes were developed primarily as markers for electron microscopy. Their very high electron density and selectivity for narrow size distributions when prepared in different ways rendered them highly suitable for ultrastructural applications [2]. The widespread use of gold labeling for light microscopy was made possible by the introduction of autometallographic enhancement methods. In these processes, the bound gold particles are exposed to a solution containing metal ions and a reducing agent; they catalyze the reduction of the ions, resulting in the selective deposition of additional metal onto the particles. On the molecular level, the gold particles are enlarged up to 30 to 100 nm in diameter; on the macroscale level, this results in the formation of a dark stain in regions containing bound gold particles, greatly increasing visibility and contrast [3]. 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
The applications of colloidal gold have been described elsewhere [4, 5]; this chapter focuses on the use of covalently linked cluster complexes of gold and other metals. A gold cluster complex is a discrete molecular coordination compound comprising a central core, or “cluster” of electron-dense metal atoms, ligated by a shell of small organic molecules (ligands) that are linked to the metal atoms on the surface of the core. This structure gives clusters several important advantages as labels. The capping of the metal surface by ligands prevents nonspecific binding to cell and tissue components, which can occur with colloidal gold [6]. Cluster compounds are more stable and can be used under a wider range of conditions. Unlike colloidal gold, clusters do not require additional macromolecules such as bovine serum albumin or polyethylene glycol for stabilization, and the total size of the label is therefore significantly smaller. Because the clusters considered in this chapter are generally less than 3 nm in diameter, this allows the preparation of probes that are much smaller than conventional immunocolloids, and cluster labeling can take advantage of the higher resolution and penetration available with smaller conjugates. Most importantly,
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Molecular Morphology in Human Tissues: Techniques and Applications while colloidal gold is adsorbed to its conjugate probe, clusters are conjugated by chemically specific covalent cross-linking. Therefore, the range of possible conjugate targeting agents includes any probe containing an appropriate reactive group. Clusters conjugates have been prepared with a wide variety of molecules that do not form colloidal gold conjugates, including lipids, oligonucleotides, peptides, and other small molecules [7]. In addition to the development of gold cluster labeling technology, this chapter also reviews new developments in the related metallographic, or metal deposition, methods. This includes gold enhancement, in which gold rather than silver is selectively deposited onto gold particles. We also describe some results obtained using another novel metallographic procedure, enzyme metallography, in which metal is directly deposited from solution by an enzymatic reaction. Because the original, and most widespread, use of metal cluster labels is in electron microscopy, many of the light microscopy methods described were developed as extensions of, or complements to electron microscopy methods, and demonstrate their greatest advantages when used with electron microscopy; therefore, reference will also be made to the electron microscope methods used in the same studies, and the unique information that can be obtained from the correlation of both methods. 4.2 CLUSTER LABELING METHODS AND RELATED TECHNOLOGIES 4.2.1
Metal Cluster Labels
The prototype gold cluster label is the undecagold cluster, which contains a core of 11 gold atoms coordinated by seven tris(aryl)phosphine ligands and three 82
halides or pseudohalides; this is then linked to biological macromolecules or other targeting agents by means of a single reactive substituent on one of the coordinated phosphine ligands [8]. However, undecagold is very small and difficult to visualize microscopically. Much better results have been obtained using the larger, 1.4-nm Nanogold label [7, 9, 10]. Although small and faintly colored in comparison with larger colloidal gold, when Nanogold is combined with autometallography, it is transformed into one of the most sensitive visualization and detection methods available. Nanogold combined with silver or gold enhancement has been used to visualize single copies of target genes in in situ hybridization experiments [11–13] and can detect as little as 0.1 pg (picogram) of a target IgG on immunodot blots [8]. While these sensitivities are now equal to or greater than those of colloidal gold, Nanogold conjugates also penetrate much more deeply into tissue sections and access hindered antigens much more effectively. Labeling has been observed at depths of up to 40 microns in tissue sections [14, 15]. This makes gold cluster labeling useful for several methods in which colloidal gold has previously produced poor results. 4.2.2 Autometallography: Silver Enhancement “Autometallography” is also called “electroless deposition,” “silver (or gold) enhancement,” or “silver (or gold) development.” It refers to the use of metal nanoparticles as “seeds” or nucleation centers that, under the right conditions, cause metal ions in solution to be reduced to metal in the zero oxidation state and deposit in layers on the seed particle. This can be extremely useful because the seed particle now becomes larger and more
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Gold Cluster Labels and Related Technologies in Molecular Morphology detectable, thus improving sensitivity. Applications include making small gold nanoparticle immunoprobes visible by EM [16] and LM [17], detection in gene or other arrays [18], detection in gels and blots [19], in situ hybridization [11–13], sensitive diagnostic tests [20, 21], and preparation of larger nanoparticles [22]. Methods of detection of metal particles are likewise expansive, including simple light absorption, electron and x-ray scattering, Raman spectral enhancement [23], interaction with fluorophores (quenching [24] and enhancement [25]). And because the metal is conductive, other properties can be utilized, such as changes in conduction or capacitance between electrodes [26]. The metals deposited can also be varied; and although silver enhancement is commonly used, gold, copper, nickel, platinum, and many other metals can be used that may be advantageous for particular applications. Electroless deposition is used to coat plastics with metals, prepare computer hard disks, and plate metals, such as silverware. How does it work? The process is somewhat related to photography, where a silver halide crystal defect caused by light becomes a nucleation site for silver reduction. In the case of a metal nanoparticle, the metal surface is the nucleation site. A developing solution is required, minimally consisting of the metal ions (e.g., Ag+) and a reducing agent (e.g., hydroquinone). The reactions are shown in Figure 4.1. The electromotive potentials for these reactions are listed on the right, and the positive sum (+0.101) indicates it is a spontaneous reaction. However, these potentials assume standard conditions of concentration, and the potentials vary with pH. At lower pH, the reaction is very slow. The metal particle surface is presumably catalytic by deforming the hydroquinone to reduce the activation energy and increase the rate of reac-
tion. What size particle is necessary? It appears that four metal atoms are sufficient [27]. At the other end of the size range, what is the largest size? Of course, the percent change in particle diameter with large particles is less for the same deposition rate, so upon enhancement, the small particles appear to “catch up” with the larger ones, leading to a more homogeneous size distribution, in some cases ∼80 nm. What are some of the drawbacks of this system? 4.2.2.1
Light Sensitivity
Some silver salts are light sensitive (e.g., silver lactate) and development should be in the dark, whereas silver acetate appears to be rather light insensitive [28]. Although some light sensitivity has been observed, useful reactions can be carried out in room lighting. 4.2.2.2
Inhomogeneous Particle Sizes
Gold nanoparticles usually have some organic coating, or are bound to biomolecules, and giving variable catalytic surfaces leading to different rates of deposition. Microscopically, some particles do not even develop at all. Additives to the developing mix, such as gum Arabic, and the speed of
2Ag
+
+ 2e
-
2Ag
0
E0 =0.7996
Hydroquinone (HQ) OH
O + H2 + 2e
OH
O
E0 = –0.699
Figure 4.1 Mechanism of silver enhancement reaction, showing half-cell potentials.
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Molecular Morphology in Human Tissues: Techniques and Applications the development affect the final size distribution. 4.2.2.3
Autonucleation
The enhancement solution by itself will form nanoparticles that are then catalytic for further growth. This is due to the exothermic reaction conditions and nucleating impurities such as small particles. This process can be delayed by various additives, or purification of reagents. It also creates a window for specific enhancement because this nonspecific metal deposition will ultimately ruin any specific reaction. One must be careful not to allow the enhancement go too long into this autonucleation zone. A useful method to obtain some additional sensitivity without autonucleation is to apply the developer for a period of time shorter than the autonucleation time, rinse it off, and then apply freshly mixed developer, which starts a new autonucleation cycle. This can be repeated several times. Because developers will eventually autonucleate, they should be used immediately after mixing their components. 4.2.2.4
Halides
Silver ions precipitate with halides, so thorough washing with deionized water is required before enhancement. 4.2.2.5
Temperature
Higher temperatures usually lead to more rapid autonucleation times and too short a window for best use. 4.2.3
Gold Enhancement
Although silver enhancement has traditionally been the most popular developer 84
for gold particles, gold enhancement [29, 30] has a number of advantages, including: (1) the pH is near 7, whereas many silver developers operate at ~pH 3.5; (2) the size distribution of the enhanced particles is sometimes narrower than with silver; (3) the product is gold, which is much better for backscatter detection by SEM or other applications [31]; (4) the gold product is chemically inert and not dissolved by oxidizing agents; for example, osmium tetroxide can dissolve silver but not gold [32]; (5) the reaction is more selective — for example, gold enhancement can be carried out in cells cultured on metal substrates [32]; and (6) the reaction can be used in the presence of chloride ions, useful for biology; silver ions form a precipitate with chloride but gold ions do not. Gold and silver enhancers are available from Nanoprobes, Inc. (www.nanoprobes.com). 4.2.4 Combined Fluorescent and Gold Probes The control that covalent labeling provides over probe architecture and configuration has led to the development of another novel class of reagents: combined fluorescent and gold probes. Selective coupling of the gold cluster to a unique site in an antibody fragment, such as a hinge thiol, allows the attachment of a fluorescent label elsewhere on the antibody via a second cross-linking reaction, to yield a probe with both fluorescent and gold labels. Combined fluorescent and gold probes, available commercially as “FluoroNanogold” (Nanoprobes, Yaphank, NY), can be used for correlative fluorescence and electron microscopy [33–37], or for checking labeling by fluorescence microscopy before undertaking electron microscopy processing.
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Gold Cluster Labels and Related Technologies in Molecular Morphology An important consideration when designing such probes is fluorescence resonance energy transfer. The overlap between the emission spectrum of the fluorescent label, and the absorption spectrum of the gold particle allows for significant nonradiative energy transfer from the fluorescent label to the gold cluster, and reduces fluorescence intensities, as described by Förster [40]. Fortunately, this process is highly dependent upon the separation of the two labels; provided they are positioned far enough apart, enough fluorescent emission remains for microscopic use. Calculations with the Nanogold cluster yield a Förster distance (the separation at which 50% of the native fluorescence is retained) of approximately 6 nm, allowing usable fluorescence when both labels are linked to a single antibody. However, extinction coefficients, and therefore overlap integrals and Förster distances, increase significantly for larger colloidal gold particles. Förster calculations indicate, for example, that for fluorescein and a 20-nm colloidal gold particle, the Förster distance is about 25 nm [41]. Although a few reports have described the preparation of combined fluorescent and colloidal gold probes, recent more rigorous studies using ultracentrifugation and pelleting to separate the gold conjugate from dissociated antibody confirmed that almost complete quenching of up to 99% or more occurs in combined 6and 18-nm colloidal gold and fluorescent conjugates: fluorescence was only obtained from the supernatent, confirming that the fluorescence signal actually arises from fluorescently labeled probes that have dissociated from the gold particles. Conjugation of the fluorescent label, in this case an Alexa Fluor 488, to a second antibody that binds to the gold-conjugated one, increases the gold–fluorophore separation to 10 to 20 nm; although quenching was still present, sufficient fluorescence was restored for observation by CCD camera, confirm-
ing the relationship between fluorescence quenching and separation [42]. 4.2.5
Nanogold Autometallography
Aside from the general procedures and properties of silver and gold enhancement of metal particles, there are more specific considerations and advantages to using small Nanogold particles as nucleating centers for the deposition reactions. The 1.4nm-diameter Nanogold (diameter of solid gold core; 2.7 nm total diameter with phosphine shell), is considerably smaller than the commonly used 10- to 40-nm gold. The advantages of using a smaller nanoparticle include (1) better penetration into tissue, (2) less steric hindrance, so that more epitopes are labeled, (3) the linkage of Nanogold to antibody is covalent and more stable than antibodies just adsorbed to 10-nm gold, (4) better washing of unbound gold, (5) there may be less background binding, and (6) small ligands, peptides, or compounds may be stably attached to the gold [9, 10], whereas small molecules usually do not adsorb stably to colloidal gold. The smaller gold particles are not as visible initially, but after enhancement they produce a clear signal, either at the EM, LM, or unaided eye level. So, after enhancement of a 10-nm colloidal gold immunoprobe vs. a Nanogold immunoprobe, which gives the strongest signal? It is our experience, using dot blots as a test, that the Nanogold conjugate, although weaker without enhancement, is more sensitive by an order of magnitude or more after they are both enhanced with silver or gold. Many studies have now been published using Nanogold for immunolocalization. Gold is the preferred marker for electron microscopy (EM), so Nanogold is ideal for that field. However, with further metal 85
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Molecular Morphology in Human Tissues: Techniques and Applications enhancement, the advantages of using small covalent gold can be appreciated in tissue applications at the LM level. Takizawa and Robinson [14] showed that Nanogold-Fab′ probes penetrated fully into 2-µm cryosections, whereas 5-nm colloidal gold hardly penetrated, and 10-nm colloidal gold-IgG probes did not enter at all; 2-µm cryosections were recut perpendicularly so that they could be viewed from the side to see the extent of penetration. Due to the far denser labeling, intense staining was observed with Nanogold. They noted that DAF, a relatively low-abundance protein in neutrophils, was difficult to detect with 10nm gold, but readily visualized with Nanogold-Fab′ . Because the Nanogold particle is so small (1.4 nm), for most applications (except high deposition or high resolution EM), it must to be silver or gold enhanced for best visibility. Detection of cellular components can be done using a directing moiety that binds to the desired target. The targeting moiety can be an antibody, antibody fragment, peptide, protein, nucleic acid, drug, carbohydrate, lipid, charged/hydrophobic/ hydrophilic group, or other ligand. Nanogold has a monofunctional linking arm that provides standard covalent crosslinking chemistry to attach to amines or thiols, or other groups if required. The smallest antibody probe giving excellent penetration was formed by linking Nanogold to recombinant Fv antibody fragments [43]. Several examples of enhanced gold in studies of human tissues are provided in the review by Lackie [44].
4.2.6
Double Labeling
Nanogold can be used in double labeling experiments, so that two labels can be visualized together. For electron microscopy, two strategies are as follows. One is to silver enhance Nanogold, but make it distinguishable from some standard colloidal gold sizes. This was done by Takizawa and Robinson [14] where Nanogold, because of its better sensitivity over colloidal gold, was used to detect a low abundance epitope. After silver enhancement, a 10-nm colloidal gold immunoconjugate was used to label the second target. Two other examples used Nanogold in double labeling with 30- and 40-nm colloidal gold in neurological tissue to distinguish receptors [45, 46]. A second strategy is to silver develop the first target gold particle to larger size, and then label the second target with gold and apply a second silver enhancement [47]. Because the first gold particles will then have been developed twice, they become distinguishably larger than the second label. 4.2.7
Enzyme Metallography
Oxidoreductases such as horseradish peroxidase are commonly used to generate colors for detection (e.g., soluble NBT/BCIP or insoluble DAB). Tissue immunohistochemistry frequently relies on the DAB reporter. A more recent alternative developed by Nanoprobes is the enzymatic deposition of gold nanoparticles that serve as substrates [48]. These produce intense metal deposits that enhance viewing and also extend the utility to EM, where the metals are clearly visible, whereas organic deposits such as DAB are not. More recently, it has been found that redox enzymes, in particular horseradish
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Gold Cluster Labels and Related Technologies in Molecular Morphology peroxidase (HRP), can also catalyze the selective deposition of metal from solution. Further investigation revealed that this reaction could be used for highly specific and sensitive staining, both for immunohistochemistry and in situ hybridization [49], and in some cases produced cleaner, darker staining than that using gold cluster labeling with autometallography [50]. Preliminary results indicate that this method, like cluster labeling, also produces specific staining in the electron microscope, and thus may be applicable as a correlative light and electron microscopic stain [50]. An interesting observation about this technology is that the deposits are very well localized, whereas DAB is known to
spread away from the site of origination before becoming fixed, leading to a lower resolution signal. Immunohistochemical and in situ hybridization detection using enzyme metallography are shown in Figure 4.2. 4.3 SPECIFIC GOLD CLUSTER PROBES AND METHODS 4.3.1 LI Silver Enhancement of Nanogold Conjugates in Light Microscopy If aldehyde-containing reagents have been used for fixation, these must be quenched before labeling. This can be
Figure 4.2 (Color Figure 4.2 follows page 106.) Intraductal comedo-carcinoma of the breast, stained for HER2 using polyclonal c-erb-B2 primary antibody, followed by EnVision secondary polymer-antibody-HRP conjugate, developed with (a) DAB (H and E counterstain) or (b) enzyme metallography (Methyl Green counterstain) (bar = 10 m); (c) HER2 in situ hybridization in non-amplified tissue, using hapten-labeled probe reagents with hematoxilin counterstain (400X original magnification); (d) HER2 highly amplified tissue, using hapten-labeled probe reagents with hematoxilin counterstain. (400X original magnification)
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Molecular Morphology in Human Tissues: Techniques and Applications achieved by incubating the specimens for 5 min in 50 mM glycine solution in PBS (pH 7.4); ammonium chloride (50 mM) or sodium borohydride (0.5 to 1 mg/ml) in PBS may be used instead of glycine. To prepare the developer, mix equal amounts of the enhancer and initiator immediately before use. Nanogold will nucleate silver deposition, resulting in a dark staining depending on development time. Additional steps, such as postfixing, may be used as required. Optimum results should be obtained using the buffers and washes specified in the instructions for the Nanogold reagents.
10. Rinse with deionized water (2 × 5 min). 11. The specimen can now be stained, if desired, before examination, with the usual reagents.
1.
PBS Buffer:
2.
3. 4. 5.
6. 7. 8. 9.
88
Spin cells onto slides using Cytospin, or use paraffin section. Incubate with 1% solution of bovine serum albumin in PBS (PBS-BSA) for 10 min to block nonspecific protein binding sites. Incubate with primary antibody, diluted at usual working concentration in PBS-BSA (1 hr or usual time). Rinse with PBS-BSA (3 × 2 min). Incubate with Nanogold reagent diluted 1/40 to 1/200 in PBS-BSA with 1% normal serum from the same species as the Nanogold reagent, for 1 hr at room temperature. Rinse with PBS (3 × 5 min). Postfix with 1% glutaraldehyde in PBS at room temperature (3 min). Rinse with deionized water (3 × 1 min). Develop specimen with freshly mixed developer for 5 to 20 min. More or less time can be used to control the intensity of the signal. A series of different development times can be used to find the optimum enhancement for your experiment; generally, a shorter antibody incubation time will require a longer silver development time.
PBS-BSA Buffer: 20 mM phosphate 150 mM NaCl pH 7.40 0.5% BSA 0.1% gelatin (high purity) Optional, may reduce background: 0.5 M NaCl 0.05% Tween 20 20 mM phosphate 150 mM NaCl pH 7.40 4.3.2 Silver Acetate Autometallography of Nanogold Conjugates for LM Nanogold-Silver Immunostaining Silver acetate autometallography [28] has been found to be highly sensitive and specific for in situ hybridization with Nanogold. Detailed protocols for specific procedures using this method are available online from the Research Institute for Frontier Questions of Medicine and Biotechnology in Salzburg, Austria (http://www.med-grenzfragen.at). Solutions A and B should be freshly prepared for every run. To prepare the enhancement solution: just before use, mix solution A with solution B. 1. 2. 3. 4.
Deparaffinize sections and bring to water through graded alcohols. Distilled water (>3 min). Antigen retrieval as appropriate. Oxidize in Lugol’s iodine solution (Merck) (5 min).
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Gold Cluster Labels and Related Technologies in Molecular Morphology 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16.
17.
Rinse in tap water (>10 sec), then distilled water (>10 sec). Reduce in 2.5% sodium thiosulfate solution until colorless (a few seconds). Wash thoroughly in tap water (>10 sec), then in distilled water (2 min). Drain off, wipe-dry area around section, and apply DAKO-pen. Immerse in PBS containing detergent (>5 min). Apply 1:20 normal serum of the species providing the secondary antibody (5 min). Drain and incubate with appropriately diluted primary antibody (overnight at 4°C). Wash in PBS-gelatin, also containing detergent (3 × 5 min). Incubate with biotinylated anti-rabbit or anti-mouse immunoglobulins (DAKO, 1:200 in PBS) (30 min at RT). Wash in PBS-gelatin, also containing detergent (3 × 5 min). Incubate with streptavidin-Nanogold diluted 1:750 in PBS-BSA (60 min at RT). Wash in PBS-gelatin, also containing detergent (3 × 5 min). Avoid the use of metal forceps from this step on! Wash repeatedly for at least 15 min in Ultrapure water and then apply silver enhancement (silver acetate autometallography). Place the slides vertically in a glass container (preferably with about 80 ml volume and up to 19 slides; Schiefferdecker-type) and cover them with the mixture of solutions A and B. Staining intensity can be checked in the light microscope during the amplification process, which usually takes about 5 to 20 min, depending on primary antibody or nucleic acid probe concentration, incubation conditions,
and the amount of accessible antigen in question. 18. Rinse carefully. 19. Counterstain with H&E or Nuclear Fast Red, dehydrate, and mount in DPX or Permount. Avoid Eukitt. Solution A: Dissolve 80 mg silver acetate (code 85140; Fluka, Buchs, Switzerland) in 40 ml of glass double-distilled water. (Silver acetate crystals can be dissolved by continuous stirring within about 15 min.) Solution B: Dissolve 200 mg hydroquinone in 40 ml citrate buffer (below). Citrate buffer: Dissolve 23.5 g trisodium citrate dihydrate and 25.5 g citric acid monohydrate in 850 ml deionized or distilled water. This buffer can be kept at 4°C for at least 2 to 3 weeks. Before use, adjust to pH 3.8 with citric acid solution. PBS: Phosphate-buffered saline (PBS) pH 7.6 containing 0.1% Tween 20 or Triton X-100. PBS-BSA: PBS pH 7.6 containing 1% bovine serum albumin. PBS-gelatin: BS containing 0.1% fish gelatin (Sigma). 4.3.3 Gold Enhancement for Light Microscopy GoldEnhance is prepared immediately before use by mixing equal amounts of Solution A (enhancer) and Solution B 89
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Molecular Morphology in Human Tissues: Techniques and Applications (activator), followed by the Solution C (initiator) and Solution D (buffer). All these four solutions are contained in the GoldEnhance kit (Nauoprobes, Inc.). For optimum results, we recommend waiting 5 to 10 min after mixing A and B before adding C and D, although the reagent will produce successful enhancement if C and D are added immediately to up to 2 hr later. The reagents are supplied in dropping bottles for easier dispensing of small amounts. If aldehyde-containing reagents have been used for fixation, it is recommended that these be quenched before labeling. This can be achieved by incubating the specimens for 5 min in 50 mM glycine solution in PBS (pH 7.4); ammonium chloride (50 mM) or sodium borohydride (0.5 to 1 mg/ml) in PBS can be used instead of glycine. The following procedure was developed for gold enhancement of in situ hybridization specimens by Hacker et al. [13] as a modification of the Nanogold-Silver Staining procedure. It has been found to be effective for enhancement of tissue sections for light microscope observation. We have found times of 10 to 20 min give optimal results; however, this reagent is intended to function under a wide range of conditions, and different washes and development times may give better results in your application. You should follow your normal procedure up to the application of the gold conjugate; the protocol below describes the steps after this: 1.
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Incubate the sections with Nanogold or colloidal gold conjugate according to current protocols or using the buffers, concentrations, and protocols recommended for the conjugate.
Wash in PBS pH 7.6, 2 × 5 min each. Wash in PBS-gelatin pH 7.6 for 5 min. 4. Repeatedly wash in distilled water for at least 10 min altogether, the last two rinses in ultrapure water (EM-grade). 5. Prepare GoldEnhance using equal amounts of the four components (Solutions A, B, C, and D); prepare at least about 80 µl per slide. 6. Dispense Solution A (enhancer: green cap) into a clean tube or dish; add Solution B (activator: yellow cap) and mix thoroughly. 7. Wait 5 min. 8. Add Solution C (initiator: purple cap) and Solution D (buffer) and mix thoroughly. 9. Apply 1 to 2 drops (about 80 µl, sufficient to cover the specimen) to the slide. 10. Develop specimen for 10 to 20 min. More or less time can be used to control particle size and intensity of signal. 11. When optimum staining is reached, immediately stop by rinsing carefully with deionized water. 2. 3.
PBS-Gelatin Buffer: 20 mM phosphate 150 mM NaCl pH 7.6 Optional, may reduce background: 0.1% gelatin (high purity) 0.5 M NaCl 0.05% Tween 20 PBS Buffer: 20 mM phosphate 150 mM NaCl pH 7.6 To obtain an especially dark signal, gold enhancement can be repeated with a freshly mixed portion of GoldEnhance.
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Gold Cluster Labels and Related Technologies in Molecular Morphology 4.3.3.1 Notes • Development starts with addition of Solution C (initiator), so apply to sample as soon as possible after adding C and D to minimize autonucleation background. • To obtain an especially dark signal, or for further development, develop longer or gold enhancement may be revitalized with a freshly mixed portion of GoldEnhance (rinse with distilled after between applications of GoldEnhance). • The development is not highly light sensitive, so may be conducted under normal room lighting, or viewed by light microscopy. • Some users reported good development omitting the use of Solution D, but deposition times are then slower. 4.3.4 Correlative Light and Electron Microscopy Nanogold and related smaller gold labels are the preferred labels for EM due to their small size, high electron scattering, and high resolution with which they bind targets. However, because Nanogold may be silver or gold developed to produce large particles or coalesced aggregates that are visible by LM, it can be used for correlative microscopy: it is possible to carry out a single labeling experiment to label both at the EM and LM level and thus to provide a direct correlation between the two. This is extremely useful because if a light microscopic experiment is done with one probe, such as a fluorescent antibody probe, and the other with a colloidal gold conjugate, the results often have been found to be inconsistent. Discord can arise from differences in penetration, binding constants, probe purity (e.g., free antibody will alter results), efficiency of binding to antigen,
washout rates, antibody differences, and different background binding characteristics. A more direct and consistent approach is to use one optimal probe, and examine its distribution both by EM and LM, even in the same cell. This is possible using Nanogold and altering the time of enhancement. An example of correlative LM/EM with silver-enhanced Nanogold is the work by Sun et al. [15], where labeling in thick sections examined by laser scanning confocal microscopy (LSCM) was matched with EM sections. Neurons were labeled with neurobiotin or biocytin, Vibratome sectioned, stained with Nanogold-streptavidin (for EM), and Cy3streptavidin (for LM) and embedded in epon/araldite. LSCM optical sectioning was used to find interesting areas, and then these were directly thin sectioned for EM. They found the Nanogold-streptavidin reliably labeled up to 40 µm into the tissue. Takizawa and co-workers [36] also immunolocalized epitopes and found them in the same cell both by light and electron microscopy. 4.3.5 Gold Cluster Labels for Chromogenic Microarray and Biochip Detection Gene arrays and chips are becoming more popular for screening tissues and cells to discover genetic differences by comparing extracted DNA, and to find up- and down-regulation by examining mRNA. Target sequences are initially placed in spots or micro squares, the sample is applied, and hybridization is detected by a reporter. Although fluorophores are commonly used, metal nanoparticles have many advantages. The signal does not fade or change over time or with observation, as fluorescence does; and detection is simpler, not requiring fluorescent optics. Alexandre and coworkers [18] have reported that Nanogold 91
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Molecular Morphology in Human Tissues: Techniques and Applications and silver enhancement functions as a simple, low-cost colorimetric detection method for DNA microarrays: a biotinylated target, human cytomegalovirus DNA, was detected after capture using Nanogold-streptavidin followed by silver enhancement by sequential application of silver nitrate followed by hydroquinone. Signals were read using an array colorimetric workstation comprising a computer tower equipped with a CCD camera, illumination, and image analysis software. The sensitivity of this method was found to be equal to that found using Cy3 fluorescence detection, corresponding to 1 amol (attomoles) of biotinylated DNA attached on an array. The high sensitivity of visualization and optical detection enabled by autometallography also makes this method suitable for use in other optical detection systems, such as biochips. 4.3.6
Gold Cluster-Labeled Lipids
Because gold clusters or nanoparticles can be attached to almost anything (DNA, proteins, drugs, carbohydrates), why not lipids? Nanogold is a good candidate for covalently linking to lipids because it is monofunctional and provides standard chemistry for making a stable covalent conjugate. The gold particle is attached to a
suitable reactive functionality positioned in the hydrophilic head-group of the lipid: the dangling, hydrophobic tail is then free to insert into liposomes or other lipidbased structures. Nanogold conjugates are available with phospholipids or fatty acids, and the structures are shown in Figure 4.3. These molecules have been used to form monolayers on air–water interfaces, creating arrays of gold particles. Gold-decorated liposomes, or “metallosomes” can be prepared by sonication of the gold-labeled lipids, either alone or mixed with unlabeled lipids, in water, and a number of different morphologies have been found for the resulting vesicles [52]. These probes can also be used to track liposomal species in the body. Adler-Moore [53] used this to monitor delivery and action of the anti-fungal channel-forming drug, amphoterin: this is most effective in a liposomal formulation where it is incorporated into 45- to 80-nm liposomes composed of hydrogenated soy phosphatidylcholine, distearoyl phosphatidylglkycerol and cholesterol, which reduced preclinical toxicity 30-fold in rodents. Fluorescently labeled liposomes both with and without the drug were localized by fluorescence microscopy in vitro, and in sections of kidney taken from mice challenged with Can-
O C NH
CH3
Palmitoyl-Nanogold (Fatty Acid) O C CH O O 2 OO
O P O NH
CH O
C
CH3 CH 3
CH2
O
Dipalmitoyl phosphatidylethanolamine-Nanogold (DPPE - phospholipid) Figure 4.3 Structure of Nanogold-conjugated lipids.
92
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Gold Cluster Labels and Related Technologies in Molecular Morphology dida albicans and subsequently injected with liposomes. Fluorescence studies showed localization of the drug-containing liposomes to the fungal cells in the mouse kidney and penetration of the fluorescent signal into the interior of fungal cells in vitro; resonance energy transfer (RET) studies showed that the constituents of drug-bearing liposomes were dispersed upon fungal penetration, suggesting that the fungal cell wall was disrupted by the drug. This was confirmed by electron microscopy studies using liposomes spiked with a small amount of dipalmitoyl phosphatidyl ethanolamine (DPPE)-Nanogold: after a 14-hr incubation with Aspergillis fumigatus (one of the drug targets), Nanogold delivered in non-drug-bearing liposomes was almost entirely incorporated into the fungal cell wall, while the Nanogold delivered in amphoterin-loaded liposomes had entered the fungal cells and was distributed throughout the cytoplasm [53]. DPPE-Nanogold with silver enhancement was also used to label cationic liposomes and elucidate their targeting to endothelial cells in tumors and areas of chronic inflammation in mice [54].
counterstains. The punctate nature of staining means that the signals are also highly localized; while this is not significant for some light microscopy applications, it means that should the need arise for subsequent electron microscopy, the same staining can be visualized at the higher resolution obtainable with EM. Most importantly, the sensitivity of this method and modifications toward low copy number targets means that it can reliably be used to detect even genes present in only one or two copies.
4.3.7 Localization and Detection of DNA: In Situ Hybridization and In Situ PCR
Although well-defined staining could be achieved for abundant targets, such as HPV16 in CaSki cells, which contain several hundred copies, by direct in situ hybridization with a biotinylated cDNA probe followed by detection with Nanogold-streptavidin and enhancement using a silver acetate developer, for the detection of very low copy number targets, a target or signal amplification step is required. While both approaches have been used, in the systems studied most widely, target amplification, which is achieved using in situ PCR [55], was found to be less specific than signal amplification, possibly due to diffusion of the amplified targets. Therefore, most of the subsequent work has utilized signal amplification, using tyramides or similar methods.
The staining produced by gold cluster labeling has several characteristics that make it ideal for in situ hybridization. The small size of gold clusters, particularly because they do not require additional macromolecules for stabilization and do not aggregate to form larger oligomers, means that they penetrate readily into cells and tissue sections to access nuclear targets. The staining produced by autometallographically enhanced gold is black, highly opaque, and punctate, and is therefore visually distinct from commonly used
A variety of different protocols have been investigated using different haptens, signal amplification schemes, and autometallography methods to optimize and simplify this method. In the original work, Hacker [56] demonstrated consistent detection of HPV16/18 in SiHa cells, known to contain only one to a few copies of the target gene, using a biotinylated DNA probe or a fluorescein-conjugated riboprobe, the latter of which was detected using a biotinylated secondary antibody that was, in turn, detected with avidin-biotin complex and 93
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Molecular Morphology in Human Tissues: Techniques and Applications biotin-HRP followed by treatment with biotin-tyramide and ultimately detection using Nanogold-streptavidin followed by enhancement with silver acetate developer. Parallel studies carried out by the same team were simplified using biotinylated hybridization probe and HRP-streptavidin to deposit biotinylated tyramide [57]. Application of the gold enhancement process to in situ hybridization has resulted in the development of protocols that are sufficiently sensitive and reproducible to find applications in diagnostic pathology. Chromogenic in situ hybridization methods have attracted a great deal of attention recently, and a consensus has developed that they offer important advantages for the practicing pathologist. Unlike fluorescent methods, they neither require expensive fluorescence optics nor dark adaptation on the part of the user; instead, staining can be interpreted using a standard bright-field light microscope. The underlying cell and tissue morphology is visualized simultaneously with the target, yielding additional information that may be helpful for diagnosis; and the autometallographically enhanced staining is permanent. Tubbs [58] describes the development of a gold-facilitated in situ hybridization (GOLDFISH) assay for the detection of HER2 gene amplification in breast carcinoma. This method correlated very well with fluorescent in situ hybridization (FISH) in a series of 104 clinical cases [59], and a simplified interpretation procedure was developed in which positive and negative results were differentiated by the degree of obscuration of the nucleus rather than the number of spots. A number of enzyme chromogenic in situ hybridization studies of the same system have been described, and both methods are reported to yield a similar degree of accuracy [60–62]. 94
Although GOLDFISH produces excellent correlation with FISH and the simplified interpretation speeds up diagnosis, the interpretation does not fully resolve the important issue of interpreting whether “low-level” amplification is clinically significant. To do so, a cutoff value must be established for the number of gene copies that constitute genuine low-level amplification; 5 and 6 copies have been proposed. Identifying such cases unambiguously requires a return to interpretation by spot counting; fortunately, image analysis methods are now available for the automation of this process. Enzyme metallography was found to be highly effective for such studies, and also represents a further simplification of the procedure: a biotinylated probe is detected using HRP-streptavidin, then treated with the metallographic substrate. Individual gene copies were sharply distinguished, supporting spot counting both manually and potentially by automated image analysis and clearly visible against counterstains [50]. In addition, staining was found to be even cleaner and backgrounds even lower using the new method. The most recent modification of this method, SILVERFISH, includes the simultaneous assessment of the concomitant HER2 protein CB11 antibody and Fast Red K stain, thus providing an internal confirmation of protein overexpression [63]. 4.3.8 Combined Fluorescent and Gold Labeling Applications Combined fluorescent and gold probes bring light microscopy and electron microscopy together even more forcibly than cluster labels alone, because the principal reason for having both labels is to obtain information from both using a single probe. Early studies showed that cellular penetration was not reduced by the attached fluorescent label, illustrated by labeling of the SC35 pre-mRNA splicing site in HeLa cells
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Gold Cluster Labels and Related Technologies in Molecular Morphology [34]. These new probes could be imaged using many microscope modalities, allowing many combinations of data. Robinson, Takizawa, and co-workers [35] used them to label and image microtubules in human leukocytes, a system that yielded very poor and erratic labeling with colloidal gold: the microtubules could be visualized, after a brief 1- to 2-min silver enhancement, by fluorescence, standard bright-field, phase contrast, differential interference, and epifluorescence microscopy. Later studies using locator grids yielded images of the same sites by fluorescence and transmission electron microscopy, demonstrating proof of principle for correlative labeling [36]. Combined Nanogold and fluorescentlabeled Fab′ antibody probes were prepared by the initial reaction of a hinge thiol group, obtained by selective reduction of the hinge disulfide in F(ab′)2 fragments, followed by reaction with maleimidoNanogold, followed by reaction of the gold-labeled conjugate with amine-reactive fluorophores. This method was later applied to the preparation of combined Cy3 and Nanogold-Fab′ probes, which were used to image the polar tubes in microsporida by fluorescence and transmission electron microscopy [37], and recently to combined Nanogold and Alexa Fluor 488 [64] or 594 conjugates. Reconciling desired sensitivity and labeling density with control over background binding initially represented a significant challenge in the use of combined fluorescent and gold probes. Because fluorescent probes often require higher concentrations to achieve acceptable labeling than immunogold probes, finding the optimum concentration for combined fluorescent and gold labeling may require a compromise. In addition, inclusion of the fluorescent label adds can add a new hydrophobic or ionic character to the
probe, which may be a mechanism for background binding. However, a number of methods have been developed to reduce this. Spector and co-workers (private communication) found, after a comparison of commonly used buffers, that washing with sodium citrate immediately before silver enhancement reduced background signal effectively in HeLa cells: 0.02 M sodium citrate at pH 7.0 was most effective for silver enhancement using the Danscher formulation, while 0.02 M sodium citrate at pH 3.5 was most effective before application of HQ Silver (Nanoprobes) [34]. More generally, the addition of 5% nonfat dried milk, either as a blocking step before addition of the combined fluorescent and gold probe, or included in the incubation buffer in which this probe is applied, has been found to greatly reduce nonspecific fluorescence signals. Combined Nanogold and fluorescently labeled streptavidin was prepared by reaction with Mono-Sulfo-NHS-Nanogold; after isolation of the gold conjugate, a second conjugation was undertaken with an amine-reactive derivative of the fluorophore. Combined Nanogold and fluorescein, Cy3 [37] and Alexa Fluor 488 and 594 streptavidin [38, 39] have been prepared in this manner. The combined Cy3 and Nanogold labeled streptavidin was used for the for the in situ detection of HPV-16 viral DNA in CaSki and SiHa cells, using the same methods described previously for Nanogold-silver staining. Using a DNA probe with tyramide signal amplification, bright fluorescent signals were obtained in CaSki cells, which contain several hundred copies of the genetic target; however, clear signals were obtained even in SiHa cells, which contain only one to a few copies of the viral DNA, produced clear fluorescence signals. Detection was confirmed by gold enhancement followed by bright-field light microscope 95
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Molecular Morphology in Human Tissues: Techniques and Applications observation, which duplicated the signal localization; as a control, when the biotinylated tyramide reagent was omitted, neither fluorescent nor gold-gold bright-field signals were observed. Signal localization in CaSki cells was confirmed by electron microscopy [37]. Recently, Takizawa and Robinson [38, 39] have described the use of combined Alexa Fluor 594 and Nanogold-labeled streptavidin for correlative localization of caveolin-1 in ultra-thin cryosections of terminal villi of the human term placenta by fluorescence and immunoelectron microscopy. The use of ultrathin cryosections enabled high spatial resolution by fluorescence microscopy because there is essentially no out-of-focus fluorescence. Electron microscopy immunolabeling obtained with colloidal gold and the combined Alexa Fluor and Nanogold probe were compared using a particle counting procedure: a higher number of particles was found with silver-enhanced Alexa Fluor and Nanogold than with colloidal gold [38, 39]. Tissue was cut into small pieces, fixed in freshly prepared 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 5% sucrose for 2 hr at room temperature, then washed and embedded in 10% gelatin in the same buffer. The solidified gelatin was cut into smaller pieces and then cryoprotected by infiltration with 2.3 M sucrose in 0.1 M sodium cacodylate (pH 7.4) overnight at 4°C. Ultrathin cryosections (100-nm thickness or less) were collected on droplets of 0.75% gelatin/2.0 M sucrose or 1% methylcellulose/1.15 M sucrose, and transferred to nickel Maxtaform “finder” grids (Graticules; Tonbridge, Kent, U.K.) to facilitate relocation of specific fluorescently labeled features for EM processing. Sections were immersed in a solution containing 1% non-fat dry milk 96
and 5% fetal bovine serum in PBS (MFBS–PBS) for 15 min at 37°C to remove the sucrose and gelatin, then washed three times in PBS and incubated in MFBS–PBS with 0.05% sodium azide to block nonspecific protein binding. The grids were incubated with biotin-labeled goat anti-chicken (13 g/ml in MFBS–PBS) for 30 min at 37°C, washed in PBS for 12 min with four changes, immersed in MFBS–PBS, and then incubated with Alexa Fluor 594 FluoroNanogold-streptavidin (diluted 1:50 in MFBS–PBS) for 30 min at room temperature. The grids were then washed in PBS for 15 min with five changes and mounted on a glass microscope slide in PBS containing 1% N-propylgallate and 50% glycerol, pH 8.0, to retard photobleaching, and coverslipped. After optical microscopy and noting regions of interest on the “finder” grid for relocation in the electron microscope, the temporary slide preparations were then disassembled and the grids washed in PBS with five changes over 15 min. The ultrathin cryosections were then fixed in 2% glutaraldehyde in PBS for 30 min, washed in distilled water for 6 min with four changes, and dried with filter paper. The grids were then floated on drops of distilled water and subsequently on drops of 50 mM 2-[N-morpholino]ethanesulfonic acid buffer, pH 6.15, for 4 min with two changes, followed by silver enhanced using a freshly prepared N-propylgallate-based silver enhancement formulation for 3 min. The same regions examined by fluorescence microscopy were relocated and electron micrographs collected. Schroeder-Reiter and colleagues [65] have reported high-resolution detection and localization of nuclear features by correlative fluorescence and scanning electron microscopy. Phosphorylated histone H3 at serine 10 in mitotic barley chromosomes isolated and mounted either on lasermarked glass slides or on standard glass
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Gold Cluster Labels and Related Technologies in Molecular Morphology slides. Slides were incubated in PBS, blocked with 1% bovine serum albumin in PBS for 30 min, and then incubated with primary antibody (polyclonal rabbit antibody against histone H3 phosphorylated at serine position 10) diluted 2:500 in the blocking solution for 1 hr; after washing in PBS, the labeled secondary antibody was applied for 1 hr. The slides were subsequently washed in PBS, and specimens postfixed with 2% glutaraldehyde in PBS. Immunogold-labeled specimens were washed with distilled water, enhanced either with GoldEnhance or HQ Silver, and then washed in 100% acetone and critical point dried. Slides were first controlled with LM in phase contrast mode, then carboncoated by evaporation to a layer of 3 to 5 nm and examined at an accelerating voltage of 12 to15 kV. Back-scattered electrons (BSE) were detected with a YAG-type detector (Autrata); secondary electron (SE) and BSE images were recorded simultaneously. Nanogold, FluoroNanogold, and 10nm colloidal gold secondary immunoprobes were compared: while a 10-nm colloidal gold conjugate gave poor labeling and lack of correlation with fluorescent signals, both Nanogold and FluoroNanogold produced dense labeling that correlated well with both fluorescence labeling and known target distribution. In addition to the combined Nanogold and fluorescently labeled immunoprobes and streptavidin, a number of other combined fluorescent and gold labeled probes have been reported. Antibody Fab′ fragments labeled with 1.8-nm platinum clusters and either fluorescein or Texas Red were found to give detectable labeling of red blood cells; [66] Robinson et al.’s [36] finding that fluorescence is still visible even after brief silver enhancement of the combined fluorescein and Nanogold probes confirms that larger cluster labels may feasibly be used to prepare combined fluores-
cent and gold probes. More recently, Texas Red and Mono-Sulfo-NHS-Nanogold were conjugated to a 10,000 MW amino-functionalized dextran; the resulting combined fluorescent and gold-tagged entity was used as a neuronal trace were used to retrogradely label spinal motor neurons innervating a median unpaired fin, the sexually dimorphic anal fin musculature in female and male Western Mosquitofish, Gambusia affinis affinis, in order to assess sex differences in spinal motor nuclei organization. The sexually dimorphic anterior transposition of the median unpaired fin, specifically the anal fin, provides a versatile experimental model for studying intercellular mechanisms, processes, and interactions during post-embryonic development, especially changes of the nervous system as the animal changes from a non-internal fertilizing to an internal fertilizing species. Retrograde tract tracing using this and other probes revealed a unique spinal cord region associated with the 12th through 14th vertebrae, a portion of the unique ano-urogenital region, containing a population of secondary motor neurons with extensive dendritic arborization; the female G. a. affinis was shown to have fewer and smaller secondary motor neurons than did males, and the neurons branching and dendritic arborization were more reduced than those in males [67]. References 1. 2.
3.
Ackerley, C.A., unpublished results, 2000. Handley, D.A., The development and application of colloidal gold as a microscopic probe, in Colloidal Gold: Principles Methods and Applications, Hayat, M.A., Ed., Academic Press, San Diego, 1989, Vol. 1, chap. 1, 1–12. Danscher, G., Autometallography. A new technique for light and electron microscopic visualization of metals in biological tissues (gold, silver, metal sulphides and metal selenides), Histochemistry, 81, 331, 1984.
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De Mey, J., Colloidal gold probes in immunocytochemistry, in Immunocytochemistry — Practical Applications in Pathology and Biology, Polak, J.M. and Van Norden S., Eds., Wright, Bristol, U.K., 1983, 82. Bendayan, M., Worth its weight in gold, Science, 291, 1363, 2001. Behnke, O. et al., Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry, Eur. J. Cell Biol., 41, 326, 1986. Hainfeld, J.F., Powell, R.D., and Hacker, G.W., Nanoparticle molecular labels, in Nanobiotechnology, Mirkin C.A. and Niemeyer, C.M., WileyVCH, Weinheim, Germany, 2004, chap. 23, 353–386. Hainfeld, J.F., Undecagold-antibody method, in Colloidal Gold: Principles Methods and Applications, Hayat, M.A., Ed., Academic Press, San Diego, 1989, Vol. 2, chap. 21, 413–429. Hainfeld, J.F. and Furuya, F.R., A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177, 1992. Hainfeld, J.F. and Powell, R.D., New frontiers in gold labeling, J. Histochem. Cytochem., 48, 471, 2000. Tubbs, R.R. et al., Supersensitive in situ hybridization by tyramide signal amplification and Nanogold silver staining: the contribution of autometallography and catalyzed reporter deposition to the rejuvenation of in situ hybridization, in Gold and Silver Staining: Techniques in Molecular Morphology, Hacker G.W. and Gu, J., Eds., CRC Press, Boca Raton, FL, 2002, chap. 9, 127–144. Tubbs, R. et al., Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of HER-2/neu gene amplification, Am. J. Pathol., 160, 1589, 2002. Hacker, G.W. et al., In situ localization of DNA and RNA sequences: super-sensitive in situ hybridization using streptavidin-Nanogold-silver staining: Minireview, protocols and possible applications, Cell Vision, 4, 54, 1997. Takizawa, T. and Robinson, J.M., Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections, J. Histochem. Cytochem., 43, 1615, 1994.
15. Sun, X.J., Tolbert, L.P., and Hildebrand, J.G., Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy, J. Histochem. Cytochem., 43, 329, 1995. 16. Scopsi, L., Silver-enhanced colloidal gold method, in Colloidal Gold: Principles Methods and Applications, Hayat, M.A., Ed., vol. 1, chap. 9, 252–297. 17. Hacker G.W. et al., Silver staining techniques, with special references to the use of different silver salts in light- and electron microscopical immunogold-silver staining, in Immunogold-Silver Staining: Principles, Methods and Applications, M.A. Hayat, Ed., CRC Press, Boca Raton, FL, 1995, chap. 2, 20–45. 18. Alexandre, I. et al., Colorimetric silver detection of DNA microarrays, Anal. Biochem., 295, 1, 2001. 19. Hainfeld, J.F. and Furuya, F.R., Silver enhancement of Nanogold and undecagold, in Immunogold-Silver Staining: Principles, Methods and Applications, Hayat, M.A., Ed., CRC Press, Boca Raton, FL, 1995, chap. 5, 71–95. 20. Chandler, J., Gurmin, T., and Robinson, N., The place of gold in rapid tests, IVD Technology, 2000 (March), 37. 21. Rocks, B.F., Bertram, V.M., and Bailey, M.P., Detection of antibodies to the human immunodeficiency virus by a silver-enhanced gold-labelled immunosorbent assay, Ann. Clin. Biochem., 27, 114, 1990. 22. Albrecht, R.M. and Meyer, D.A., Size selective synthesis of colloidal platinum nanoparticles for use as high resolution EM labels, Microsc. Microanal., 8 (Suppl 2: Proceedings), Lyman, C.E. et al., Eds., Cambridge University Press, New York, 2002, 124. 23. Grubisha, D.S. et al., Femtomolar detection of prostate-specific antigen: an immunoassay based on surface-enhanced Raman scattering and immunogold labels, Anal. Chem., 75, 5936, 2003. 24. Dulkeith, E. et al., Fluorescence quenching of dye molecules near gold nanoparticles: radiative and nonradiative effects, Phys. Rev. Lett., 89, 203002, 2002. 25. Malicka, J., Gryczynski, I., and Lakowicz, J.R., DNA hybridization assays using metal-enhanced fluorescence, Biochem. Biophys. Res. Commun., 306, 213, 2003.
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Gold Cluster Labels and Related Technologies in Molecular Morphology 26. Moller, R. et al., DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold, Nucleic Acids Res., 28, E91, 2000. 27. Hamilton, J.F. and Logel, P.G., The minimum size of silver and gold nuclei for silver physical development, Photogr. Sci. Eng., 18, 507, 1974. 28. Hacker, G.W. et al., Silver acetate autometallography: an alternative enhancement technique for immunogold-silver staining (IGSS) and silver amplification of gold, silver, mercury and zinc tissues, J. Histotechnol., 11, 213, 1988. 29. Hainfeld, J.F. and Powell, R.D., Silver- and goldbased autometallography of Nanogold, in Gold and Silver Staining: Techniques in Molecular Morphology, Hacker, G.W. and Gu, J., Eds., CRC Press, Boca Raton, FL, 2002, 29–46. 30. Hainfeld, J.F. et al., Gold-based autometallography, Microsc. Microanal., 5 (Suppl 2: Proceedings), Bailey, G.W. et al., Eds., Springer-Verlag, New York, 1999, 486. 31. Hainfeld, J.F. and Powell, R.D., Silver- and goldbased autometallography of Nanogold, in Gold and Silver Staining: Techniques in Molecular Morphology, Hacker, G.W. and Gu, J., Eds., CRC Press, Boca Raton, FL, 2002, chap. 3, 29–46. 32. Owen, G.R. et al., Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions, Cell Biol. Int., 25, 1251, 2001. 33. Powell, R.D. and Hainfeld, J.F., Combined fluorescent and gold probes for microscopic and morphological investigations, in Gold and Silver Staining: Techniques in Molecular Morphology, Hacker, G.W. and Gu, J., Eds., CRC Press, Boca Raton, FL, 2002, chap. 7, 107–116. 34. Powell, R.D. et al., A covalent fluorescent-gold immunoprobe: “simultaneous” detection of a premRNA splicing factor by light and electron microscopy, J. Histochem. Cytochem., 45, 947, 1997. 35. Robinson, J.M. and Vandré, D.D., Efficient immunocytochemical labeling of leukocyte mikrotubules with FluoroNanogold: an important tool for correlative microscopy, J. Histochem. Cytochem., 45, 631, 1997. 36. Takizawa, T., Suzuki, K., and Robinson, J.M., Correlative microscopy using FluoroNanogold on ultrathin cryosections. Proof of principle, J. Histochem. Cytochem., 46, 1097, 1998.
37. Powell, R.D. et al., Combined Cy3/Nanogold conjugates for immunocytochemistry and in situ hybridization, Microsc. Microanal., 5 (Suppl 2: Proceedings), Bailey, G.W. et al., Eds., SpringerVerlag, New York, 1999, 478. 38. Takizawa, T. and Robinson, J.M., Correlative microscopy of ultrathin cryosections is a powerful tool for placental research, Placenta, 24, 557–565, 2003. 39. Takizawa, T. and Robinson, J.M., Ultrathin cryosections. An important tool for immunofluorescence and correlative microscopy, J. Histochem. Cytochem., 51, 707, 2003. 40. Förster, Th., Zwisschenmolekulare Energie-wandung und Fluoreszenz, Ann. Physik, 2, 55, 1948. 41. Powell, R.D. et al., Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy, Microsc. Res. Tech., 42, 2, 1998. 42. Kandela, I.K. et al., Fluorescence quenching by colloidal heavy metals: implications for correlative fluorescence and electron microscopy studies, Microsc. Microanal., 9 (Suppl 2: Proceedings), Piston, D. et al., Eds., Cambridge University Press, New York, 2003, 1194CD. 43. Ribrioux, S. et al., Use of nanogold- and fluorescent-labeled antibody Fv fragments in immunocytochemistry, J. Histochem. Cytochem., 44, 207, 1996. 44. Lackie, P.M., Immunogold silver staining for light microscopy, Histochem. Cell Biol., 106, 9, 1996. 45. Matsubara, A. et al., Organization of AMPA receptor subunits at a glustamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti, J. Neurosci., 16, 4457, 1996. 46. Nusser, Z. et al., Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method, J. Neurosci., 15, 2948, 1995. 47. Yi, H. et al., A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level, J. Histochem. Cytochem., 49, 279, 2001. 48. Mayer, G. et al., Introduction of a novel HRP substrate-nanogold probe for signal amplification in immunocytochemistry, J. Histochem. Cytochem., 48, 461, 2000.
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Molecular Morphology in Human Tissues: Techniques and Applications 49. Hainfeld, J.F. et al., Enzymatic metallography: a simple new staining method, Microsc. Microanal., 8 (Suppl 2: Proceedings), Voekl, E. et al., Eds., Cambridge University Press, New York, 2002, 916CD. 50. Tubbs R. et al., Enzyme metallography (EnzMet): a novel second generation bright field assay for detection of HER2 gene amplification in breast carcinoma, J. Mol. Diagn. (AMP 2003 Meeting Abstracts), 5, 279, 2003. 51. Furuya, F.R. et al., unpublished results, 2003. 52. Hainfeld, J.F., Furuya, F.R., and Powell, R.D., Metallosomes, J. Struct. Biol., 127, 152–160, 1999. 53. Adler-Moore, J., AmBisome targeting to fungal infections, Bone Marrow Transplantation, 14, S3, 1994. 54. Thurston, G. et al., Cationic liposomes target endothelial cells in tumors and chronic inflammation in mice, J. Clin. Invest., 101, 1401, 1998. 55. Hacker, G.W. et al., High-performance Nanogold in situ hybridization and in situ PCR, Cell Vision, 3, 209, 1996. 56. Hacker, G.W. et al., In situ localization of DNA and RNA sequences: super-sensitive in situ hybridization using streptavidin-Nanogold-silver staining: minireview, protocols and possible applications, Cell Vision, 4, 54, 1997. 57. Zehbe, I. et al., Sensitive in situ hybridization with catalyzed reporter deposition, streptavidinNanogold, and silver acetate autometallography. Detection of single-copy human papillomavirus, Am. J. Pathol., 150, 1553, 1997. 58. Tubbs, R. et al., Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of HER-2/neu gene amplification, Am. J. Pathol., 160, 1589, 2002. 59. Tubbs, R. et al., Interobserver interpretative reproducibility of GOLDFISH, a first generation gold-facilitated auto-metallographic bright field in situ hybridization assay for HER-2/neu amplification in invasive mammary carcinoma, Am. J. Surg. Pathol., 26, 908, 2002.
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60. Tanner, M. et al., Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples, Am. J. Pathol., 157, 1467, 2000. 61. Gupta, D. et al., Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer, Am. J. Clin. Pathol., 119, 381, 2003. 62. Arnould, L. et al., Agreement between chromogenic in situ hybridisation (CISH) and FISH in the determination of HER2 status in breast cancer, Br. J. Cancer, 88, 1587, 2003. 63. Tubbs, R., Pettay, J., Powell, R., Mele, J., DownsKelly, E., Hicks, D., and Hainfeld, J., Enzyme metallography (EnzMet™): a novel second generation bright field assay for detection of HER2 gene amplification in breast carcinoma, presented at the Association of Molecular Pathology, Orlando, November 19–21, J. Molec. Diag., 5, 270, 2003. 64. Hainfeld, J.F. et al., Combined Alexa-488 and Nanogold antibody probes, Microsc. Microanal., 8 (Suppl 2: Proceedings), Lyman, C.E. et al., Eds., Cambridge University Press, New York, 2002, 1030CD. 65. Schroeder-Reiter, E., Houben, A., and Wanner, G., Immunogold labeling of chromosomes for scanning electron microscopy: a closer look at phosphorylated histone H3 in mitotic metaphase chromosomes of Hordeum vulgare, Chromosome Res., 11, 585, 2003. 66. Powell, R.D. et al., Dual-labeled probes for fluorescence and electron microscopy, Microsc. Microanal., 8 (Suppl 2: Proceedings), Bailey, G.W. et al., Eds., Springer, New York, 1998, 992. 67. Rosa-Molinar, E. et al., Colloidal gold conjugate of recombinant cholera toxin b-subunit of Alexa Fluor and dextran-Texas Red-Nanogold fluorescent dyes for use in correlative microscopy and intravital imaging, Microsc. Microanal., 9 (Suppl 2: Proceedings), Piston, D. et al., Eds., Cambridge University Press, New York, 2003, 182.
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Gold- and Silver-Facilitated Metallographic In Situ Hybridization Procedures for Detection of HER2 Gene Amplification Raymond R. Tubbs, James Pettay, Marek Skacel, Erinn DownsKelly, Richard D. Powell, David G. Hicks, and James F. Hainfeld
5.1 INTRODUCTION Clinical laboratory assessment of amplification of the HER2 gene and immunohistochemical (IHC) detection of the HER2 encoded protein provide important information used in the management of patients with invasive carcinoma of the breast. Information regarding HER2 status is important in three key aspects: overall prognosis, predication of response to adriamyacine based regimens (and lack of response to hormone receptor therapy), and selection of patients for treatment with humanized monoclonal antibodies (Herceptin; Trastuzumab). In theory, the most appropriate information to provide for clinical management is the presence of encoded HER2 protein on the surface of tumor cells. In practice, selecting the appropriate laboratory assay for profiling HER2 is much more complicated. In this setting, the advantages of ultrasensitive antigen retrieval actually work against the pathologist, providing exquisitely sensitive detection of small 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
amounts of protein on the cell membrane. The small amounts of protein present constitutively on the cell membrane appear amplified through the power of antigen retrieval, leading to apparent false positive results using IHC. These apparent artifacts occur more frequently with polyclonal rather than monoclonal antibodies, and are most likely artifactual because RNA in situ hybridization fails to reveal amplified message in most IHC positive/fluorescence in situ hybridization (FISH) negative discordant cases [1]. Posttranslational rather than transcriptional regulation of HER2 protein expression is a possibility, although considered unlikely in view of the results of studies done with frozen section immunohistochemistry [2, 3]. Although clinical outcome studies are sparse, FISH seems to be the method of choice because the subjective intermediate levels of positivities (IHC 1+ and 2+ standing reactions) are almost never observed in FISH — the results are nonambiguously nonamplified or overtly amplified, and FISH concordance between observers is
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Molecular Morphology in Human Tissues: Techniques and Applications much higher than with IHC [1, 4–9]. Furthermore, fluorescent signals bleach upon observation and fade upon storage, whereas chromogenic detection is a permanent record, important in pathology, especially if medical decisions are based on it and the evidence must be evaluated by several pathologists, or revisited at a later date. Even if the fluorescence is photographed for a permanent record, this will be a small fraction of the slide, and later examination of the complete tissue will be compromised. However, pathologists have been slow to adopt FISH for clinical laboratory testing for HER2 gene status. Acquisition of additional expensive instrumentation, training of new personnel, and the counterintuitive aspects of “working in the dark” are probably the chief reasons for this reluctance. Consequently, new procedures using brightfield assays for direct visualization of gene amplification have been developed, and early studies are very encouraging [10–20]. Our initial experience with first-generation chromogenic in situ hybridization (CISH) systems concerned us greatly. In our experience, all amplified cases were easily and nonambiguously detected, but identification of endogenous, nonamplified signals in tumor cells and non-neoplastic cells (internal hybridization efficiency control) was inconsistent at best. For this reason, we have adopted a metallographic procedure as an alternative to conventional peroxidase-based CISH. The first generation utilized gold-facilitated autometallographic in situ hybridization (GOLDFISH), a technique that combined the robust amplification properties of catalyzed reporter deposition/tyramide signal amplification (CARD/TSA) with the deposition of metallic gold upon streptavidin-Nanogold particles [18]. Analytical validation and interobserver reproducibili102
ty have been excellent [17, 18]. The technique relied on radial signal growth attributable to both the CARD/TSA and autometallographic components of the assay, and in amplified cases produced massive black confluent signals that occupied the majority of the nucleus. The assay was specifically designed to preclude enumeration of specific signals, and worked very well for the majority of cases. However, in occasional tumors polysomic for chromosome 17 (approximately 5–10% of breast cancer cases in routine clinical practice), the property of the GOLDFISH method that worked so well for differentiation between amplified and nonamplified cases was somewhat problematic for these cases displaying polyionic states of chromosome 17. We subsequently adopted a new procedure developed by Hainfeld et al. — that of enzyme metallography — to this application [21]. The technique has several important advantages over GOLDFISH, while retaining its strengths. Specifically, conventional light microscopy can be used for scoring the slides, semi-quantitative cutoffs (which are described below) can be used to reliably score the preparations using FISH as the reference standard, and the more discrete size of the particle allows for straightforward enumeration of HER2 signals and identification of pseudo-amplifications due to polysomic states of chromosome 17. Future plans for modification of the assay include the addition of simultaneous protein detection analogous to CODFISH (concomitant oncoprotein detection with fluorescent in situ hybridization) in brightfield mode, and dual-color detection of HER2 gene and chromosome 17 centromere, for simultaneous detection of chromosome 17 polysomic states [22].
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Gold- and Silver-Facilitated Metallographic In Situ Hybridization Procedures 5.2 FIXATION AND TISSUE PROCESSING Assays for HER2 gene amplification and protein overexpression approved in the United States by the Food and Drug Administration include PathVysion (FISH: Vysis, Downers Grove, IL), HercepTest (IHC, DakoCytomation, Carpinteria, CA), and Pathway (CB11 IHC, Ventana Medical Systems, Tucson, AZ). For each of these assays, the intended use is for unstained paraffin sections of formalin-fixed tumor tissue from patients with carcinoma of the breast. No firm data is available on exactly which cases are routinely evaluated clinically for HER2 status, although anecdotal information suggests that most laboratories are testing for at least estrogen and progesterone receptor and HER2 by either IHC or FISH. The common algorithm in the United States employs a two-step process whereby IHC is used initially, followed by reflex FISH testing for IHC “2+” FDA scoring category cases. The merits of this approach are beyond the scope of this protocol description.
strong recommendation is to use formalinfixed tissue for HER2 clinical assays. For IHC, FISH, and bright field ISH testing, cell conditioning/antigen retrieval is essential for reliable detection of the target. This enzyme metallography procedure is no exception, and stripping away nucleoprotein bound to the DNA target is critical for proper exposure of the HER2 gene for hybridization. In our experience, this is best accomplished using heat-mediated cell conditioning followed by gentle protein digestion with proteinase K. The details are outlined in the procedure below. The balance, of course, is the satisfactory removal of nucleoproteins while preserving the structural detail necessary for proper use of in situ hybridization. Overdigestion of the protein results in severe loss of structural integrity and the inability to correlate the genotype with the histologic appearance of the tumor. Underdigestion precludes satisfactory exposure of the DNA target to the probe, resulting in an absence of hybridization and failure to detect the gene copy.
There is virtually no peer-reviewed literature supporting the equivalency of alternative fixatives such as Prefer for either IHC- or DNA-based clinical assays. Our
5.3 MATERIALS AND PROTOCOL PROCEDURE
Reagent Xylenes 80% Dehydrant 95% Dehydrant 100% Dehydrant Target Retrieval Solution Proteinase K Enzyme Digestion Trizma HCl 1 M pH 7.6 Hydrogen peroxide 30% Water, DNAase- Free SSC 20X NP-40 Tween-20 PBS 10X Nuclear Fast Red
Catalog Number C4330 6401 6301 6201 0S1700
City, State McGaw, IL Kalamazoo, MI Kalamazoo, MI Kalamazoo, MI Carpinteria, CA
S3004 T-2444 H-0904 W-4502 S-6639 I-3021 P-9416 70011-044 1255B
Carpinteria, CA St. Louis, MO St. Louis, MO St. Louis, MO St. Louis, MO St. Louis, MO St. Louis, MO Grand Island, MI Middleton, WI
Vendor Allegiance Richard-Allan Scientific Richard-Allan Scientific Richard-Allan Scientific DakoCytomation DakoCytomation Sigma Sigma Sigma Sigma Sigma Sigma Gibco/Invitrogen Newcomer Supply
5.3.1
Reagent Sources
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Procedure
Paraffin-embedded tissue slides are heated in a dry oven at 60°C overnight. Slides are deparaffinized with three 10-min washes of xylenes prior to two 1-min washes of 100% and 95% alcohol and then soaked in water for 5 min. Cell conditioning, probe hybridization, stringency washes, and target detection are performed according to the manufacturer’s instructions using proprietary reagents (patent pending). 5.3.3
Probe Selection
In our experience, the system functions optimally with DNA probe isolated from cosmids or other vectors, nick translated or random primer labeled with an appropriate hapten/ligand. A variety of hapten/ligand labels can be used — biotin, dinitrophenyl (DNP), digoxigenin, and fluorescence isothiocynate (FITC as immunogenic hapten, not as fluorochrome). The use of some of these probe formats may involve intellectual property issues. 5.3.4
Hybridization Conditions
The procedure outlined above has been optimized for digoxigenin labeled probe. Other haptens and probes of different sizes and base content will have varying Tm (melting temperature) values, and some empirical optimization is usually required. In our experience, although the TM for the probe can be calculated, the most practical approach is a series of classic checkerboard conditions that will usually yield the appropriate experimental conditions. Our initial GOLDFISH procedure employed catalyzed reporter deposition/tyramide signal amplification (CARD/TSA). 104
The second generation of metallographic ISH does not require CARD/TSA. Furthermore, the CARD components of the original GOLDFISH method, while resulting in radial signal growth and striking signal amplification, also produced confluent signals and precluded signal enumeration. Although the GOLDFISH approach worked well in the majority of cases, the second-generation enzyme metallography signals look very similar to FISH, readily permitting enumeration HER2 gene copy directly with conventional microscopy. Simple streptavidin-peroxidase or secondary antibody labeled to peroxidase does not produce sufficient amplification in the enzyme metallography component of the assay. Some form of polymerized peroxidase-labeled anti-Ig antibodies is necessary to achieve the proper signal amplification. Polymerized HRP antibody can be obtained from several vendors. 5.3.5 Scoring of Preparations and Interpretation The slides are evaluated with conventional bright-field microscopy using an ordinary light microscope (Figure 5.1 A through D). Oil immersion microscopy is not necessary for routine clinical use. At lower magnification, usually with a 10X objective (total magnification 100X), amplified signals are readily visible in the nuclei of infiltrating tumor cells if gene amplification is present; “high dry” using a 40× objective (400× total magnification) or 630× final magnification are entirely sufficient for evaluation of HER2 nonamplified and polysomic cases. The following semi-quantitative scoring system can be readily applied without requiring specific cell-by-cell enumeration:
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Gold- and Silver-Facilitated Metallographic In Situ Hybridization Procedures Copy Number
Interpretation 1 or 2
1–2 3–5
HER2 nonamplified Nonamplified/polysomic (pseudoamplification due to polysomic state for chromosome 17) HER2-amplified
≥6
This approach does not require cell-bycell counting of signals, but an overall semi-quantitative assessment. The semiquantitative categories above refer to the genotype in greater than 90% of the invasive carcinoma tumor cells. The designation of cases as not amplified/polysomic, due to “pseudo-amplification” attributable to polysomic states of chromosome 17, is
derived from our experience with thousands of cases where we have investigated HER2 gene copy using a two-color FISH probe set (PathVysion, Vysis). These cases are nearly always apparent low-level amplifications which, in >95% of cases, correct to normal HER2/CEP17 ratios with twocolor FISH probes.
References 1.
A
B
C
D
Tubbs, R.R. et al., Discrepancies in clinical laboratory testing of eligibility for Trastuzumab therapy: apparent immunohistochemical false-positives do not get the message, J. Clin. Oncol., 19, 2714, 2001.
Figure 5.1 (Color Figure 5.1 follows page 106.) Photomicrographs of invasive ductal carcinoma of breast, HER2 gene amplification. Enzyme metallography (1A), chromogenic ISH using diaminobenzidine (1B), dark-field microscopy of enzyme metallography preparation (1C), and dual-color FISH (1D; HER2 = red, CEP17 = green, DAPI counterstain) are illustrated.
105
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Molecular Morphology in Human Tissues: Techniques and Applications 2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
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Slamon, D.J. et al., Studies of the HER-2/Neu proto-oncogene in human breast and ovarian cancer, Science, 244, 707, 1989. Slamon, D.J., Studies of the HER-2/Neu protooncogene in human breast cancer, Cancer Invest., 8, 253, 1990. Clinical laboratory assays for HER-2/Neu amplification and overexpression: quality assurance, standardization, and proficiency testing, Arch Pathol Lab. Med., 126, 803, 2002. Pauletti, G. et al., Assessment of methods for tissue-based detection of the HER-2/Neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry, J. Clin. Oncol., 18, 3651, 2000. Perez, E.A. et al., HER-2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization, Mayo. Clin. Proc., 77, 148, 2002. Press, M.F. et al., Sensitivity of HER-2/Neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression, Cancer Res., 54, 2771, 1994. Press, M.F. et al., HER-2/Neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas, J. Clin. Oncol., 2894, 1997. Press, M.F. et al., Evaluation of HER-2/Neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens, J. Clin. Oncol., 20, 3095, 2002. Arnould, L. et al., Agreement between chromogenic in situ hybridization (CISH) and FISH in the determination of HER2 status in breast cancer, Br. J. Cancer, 88, 1587, 2003. Dandachi, N., Dietze, O., and Hauser-Kronberger, C., Chromogenic in situ hybridization: a novel approach to a practical and sensitive method for the detection of HER-2 oncogene in archival human breast carcinoma, Lab. Invest., 82, 1007, 2002. Gupta, D. et al., Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/Neu oncogene in breast cancer, Am. J. Clin. Pathol., 119, 381, 2003. Joensuu, H. et al., Amplification of erbB2 and erbB2 expression are superior to estrogen receptor status as risk factors for distant recurrence in pT1N0M0 breast cancer: a nationwide population-based study, Clin. Cancer Res., 9, 923, 2003.
14. Kumamoto, H. et al., Chromogenic in situ hybridization analysis of HER-2/Neu status in breast carcinoma: application in screening of patients for Trastuzumab (Herceptin) therapy, Pathol. Int., 51, 579, 2001. 15. Park, K. et al., Comparing fluorescence in situ hybridization and chromogenic in situ hybridization methods to determine the HER-2/Neu status in primary breast carcinoma using tissue microarray, Mod. Pathol., 16, 937, 2003. 16. Tanner, M. et al., Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/Neu oncogene amplification in archival breast cancer samples, Am. J. Pathol., 157, 1467, 2000. 17. Tubbs, R. et al., Interobserver interpretative reproducibility of GOLDFISH, a first generation gold-facilitated autometallographic bright field in situ hybridization assay for HER-2/Neu amplification in invasive mammary carcinoma, Am. J. Surg. Pathol., 26, 908, 2002. 18. Tubbs, R. et al., Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of HER-2/Neu gene amplification, Am. J. Pathol., 60, 1589, 2002. 19. van de Vijver, M., Emerging technologies for HER-2 testing, Oncology, 63(Suppl. 1), 33, 2002. 20. Zhao, J. et al., Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma, Mod. Pathol., 15, 657, 2002. 21. Hainfeld, J.F.E., Tubbs, R., and Powell, R. D., Enzymatic metallography: a simple new staining method, in Microscopy and Microanalysis 2002, 8 (Suppl. 2), Voelkl, E., Piston, D., Gauvin, R., Lockley, A.J., Bailey, G.W., and McKernan, S., Eds., Cambridge University Press, New York, 2002, 916. 22. Tubbs, R. et al., Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH): a fluorescence-based assay enabling simultaneous visualization of gene amplification and encoded protein expression, J. Mol. Diagn., 2, 78, 2000.
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Toward Molecular Sensitivity: Tyramide Signal Amplification in Molecular Morphology Gerhard W. Hacker, James Pettay, and Raymond R. Tubbs
6.1 INTRODUCTION AND BRIEF HISTORICAL OVERVIEW Biomedical scientists have always dreamed of being able to detect single molecules. In the case of single gene sequences, this dream became a reality with the introduction of the polymerase chain reaction (PCR) in vitro during the mid-1980s by Kary Mullis [1]. In the case of immunology, fulfillment of this dream has, to our knowledge, not yet been convincingly proven. Adaptation of molecular biology methods to microscopy, allowing reliable in situ detection of specific genes and other substances, has led to the new discipline of molecular morphology, a term first suggested more than a decade ago: At a series of congresses, the first of which was organized in Salzburg, Austria, in 1992, the new term was introduced. A molecular morphology journal was founded soon thereafter, Cell Vision — Journal of Analytical and Molecular Morphology (Eaton Press, Natick, MA) and subsequently fused with Applied Immunohistochemistry, now published as Applied Immunohistochemistry and Molecular Morphology (AIMM; http://www.appliedim0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
munohist.com/) (Lippincott Williams & Wilkins, Philadelphia, PA). In parallel with the first congresses explicitly dedicated to this new area, a new society was born, today called the “International Society for Analytical and Molecular Morphology” (ISAMM; http://www.isamm.org). Whereas the original methods of in situ hybridization (ISH) in the early 1980s had relied solely on radioactive labels, nonradioactive ISH protocols were introduced soon thereafter — techniques that are much less harmful to the environment and nonhazardous to the investigator [2–4]. However, at that time, radioactive labels had been regarded as the most sensitive means to detect specific gene sequences in situ — and some researchers still use them. With the development of more advanced methods of nonradioactive ISH, it became possible to greatly increase sensitivity, and biotin-labeled nucleic acid probes (Enzo, NY) in combination with streptavidin-peroxidase, alkaline phosphatase-NBT-BCIP, or colloidal gold-labeled streptavidin [5–12] allowed a relatively sensitive detection in the range of 10 to 20 copies of a specific DNA sequence per cell [10]. Some authors already may have reached single
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Molecular Morphology in Human Tissues: Techniques and Applications copy sensitivity, as has been described for the detection of Epstein Barr virus [13]; sometimes, this was also possible with an easy-to-perform gold-silver setup for the detection of human papillomavirus in SiHa cells [14]. A real breakthrough, however, was achieved by Bagasra [15] and Haase [16], who were for the first time able to utilize the PCR principle for microscopy. Genuine single-copy sensitivity for gene-detection in situ was achieved for the first time by these extraordinary U.S. scientists, thereby relying on peroxidase as the label for the reaction they called in situ PCR (ISPCR) (see Chapter 12). Soon after, in Europe, another approach was introduced for IS-PCR, one that relied on Nanogold® particles covalently labeled to streptavidin [9, 17–23] (Nanoprobes, Inc., Yaphank, NY), and the latter principle for the first time permitted the visualization of single HPV gene copies in both light and the electron microscope [24]. For the highly sensitive detection of mRNA, a different method, the in situ 3SSR reaction (“selfsustained sequence-based replication”) was published, a promising technology that unfortunately was not followed up by others, probably as a consequence of its relatively complex and laborious laboratory requirements and expense [25]. IS-PCR is based on amplification of DNA sequences within the cell. The setup described in this chapter, however, requires much less laboratory experience than ISPCR. It uses a principle in which, instead of amplifying the copy number of the nucleic acid sequence in question, the amount of label molecules/particles is maximized, also referred to as “label” or “signal amplification.” By building up a network of (biotin-)labeled tyramides at the sites of peroxidase molecules deposited in a perox108
idase-based ISH reaction executed beforehand, it became possible to detect single copies of certain HPV DNA sequences for the first time [26–29]. The basis for this approach was initially developed by Mark Bobrow [30, 31] (now at Perkin-Elmer), who published a procedure he initially termed “catalyzed reporter deposition.” Bobrow had originally suggested the use for biochemical applications. The procedure in the meantime has been patented by Perkin-Elmer/NEN and is being marketed under different names, such as “tyramide signal amplification” (TSA; Perkin-Elmer Life Sciences, http://lifesciences.perkinelmer.com), or licensed from Perkin-Elmer, as “catalyzed signal amplification” (CSA; DakoCytomation, Glostrup, Denmark). The labeling systems being utilized for tyramide-based detection methods are either fluorescent, Nanogold-silver, Nanogold-gold, or enzymes (mainly peroxidase with diaminobenzidine-tetrahydrochloride-(DAB-) – H2O2-development); (see, for example, [26, 27, 32, 33]). Tyramide-based amplification systems display a number of advantages over all earlier methods of specific DNA or RNA detection, including highest sensitivity and detection efficiency, high reliability/reproducibility, speedy performance, and relatively low cost. This chapter presents optimized protocols as currently used by the authors for nonfluorescent detection, along with detailed technical descriptions, suggested applications, and troubleshooting.
6.2 TYRAMIDE-AMPLIFIED NANOGOLD-SILVER/GOLD ISH The first system published that allowed proven single DNA copy sensitivity in the transmitted LM without the necessity of
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Toward Molecular Sensitivity PCR was a “CARD-Nanogold-silver” protocol [26–29, 34]. For a number of reasons, the authors of this chapter prefer to use modifications of this technique in contrast to using enzymes such as peroxidase. Metal-amplified Nanogold-particles result in a specific jet-black appearance very distinctly visible against the unlabeled background. This allows screening of the preparations with low-power objectives in the LM (which is a distinct advantage when used for diagnostic and scientific screening of many slides in only a short time), use of conventional counterstains such as hematoxilin and eosin (H&E), or reliable computer-based image analysis. Furthermore, it is possible to transfer the very same preparations to the transmission electron microscope (TEM) and post-analyze the localization of the specific precipitate in the section, cells, and even subcellular structures, as described by Muss in [12, 24]. The Nanogold-silver/gold principle itself originated more than two decades ago when the Danish heavy metal specialist Gorm Danscher introduced silver enhancement (autometallography, AMG) to detect catalytic tissue metals in microscopy [18, 35–39]. His technique became a prerequisite for obtaining a higher sensitivity in immunogold applications. Combining the AMG reaction with colloidal gold-labeled enzyme histochemistry and immunohistochemistry (IHC) was published simultaneously in 1983 by Danscher and Holgate et al. (U.K.), respectively. Their work is considered as a genuine breakthrough that more than 15 years later led to first verification of molecular detection sensitivity in the DNA detection field [38, 40, 41]. The original IHC technique using gold and silver was called immunogold-silver staining (IGSS) [41] and, at that time did dramatically improve sensitivity and detection effi-
ciency [18, 40–43]. Under the LM, conglomerations of gold particles embedded in silver appear as black precipitates with a distinctly sharper appearance than the reaction products of most enzyme-labeled preparations, thus offering a variety of advantages in addition to high sensitivity [18, 42, 44]. The introduction of gold crystals surrounded by organic molecules [20–23, 45] (Nanogold; Nanoprobes, Inc., Yaphank, NY; http://www.nanoprobes.com), covalently bound to streptavidin and other macromolecules, and also its combination with fluorescent labels (FluoroNanogold) furthered the utilization of IGSS in the world of diverse techniques of molecular morphology (see also Chapter 4 in this book) [12]. Parallel to the first publication of the CARD-Nanogold-silver protocol [26], a new type of heavy metal enhancement was developed, based on gold ions instead of the silver ions originally used in “silver enhancement-AMG.” The application of the GoldEnhance procedure together with TSA/CSA now yields a very smoothly proceeding, better-controlled increase of the size of the clustered gold label; contributes to greater ease in reproducing the laboratory protocol; and yields an even more distinct outcome of the staining reaction [28, 29]. Two protocols are given in the following section for tyramide- and gold-based ISH: (1) Section 6.2.1 presents a protocol slightly modified from the original [26, 27, 34], using either TSA-NEN-Perkin-Elmer reagents, or CSA reagents as marketed by DakoCytomation, in combination with Nanogold-labeled streptavidin; and (2) Section 6.2.2 describes the GOLDFISHprocedure [46, 47]. For enhancement of the gold label, the protocol for silver acetate AMG (Section 6.2.3.1.) [18] and a description of the GoldEnhance procedure are given (Section 6.2.3.2.). 109
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Molecular Morphology in Human Tissues: Techniques and Applications 6.2.1 Protocol A: TSA-NanogoldSilver/Gold ISH Modified after Zehbe et al. [26] and Hacker et al. [34], this CARD protocol is well suited for extraordinarily sensitive ISH and uses biotinylated tyramide either from Perkin-Elmer Life Sciences, or from DakoCytomation, and streptavidin-Nanogold combined with silver acetate AMG, or with gold-ion-based AMG (GoldEnhance; Nanoprobes, Inc., Yaphank, NY). In our hands, it allows a reliable detection of even single copies of DNA sequences, such as human papillomavirus (HPV) subtypes, in routinely formalin-fixed and paraffinembedded tissue specimens, as well as on formalin-fixed cytological preparations. Other DNA viruses, as well as mRNA and other RNA detections have also been tested. CARD, as developed by Bobrow et al. [30, 31], is patented by Perkin-Elmer/NEN Life Sciences Products under the term TSA (Boston, MA; http://www.nenlifesci.com) (Tyramide Signal Amplification). We have successfully used biotinylated tyramides from the “TSA-Indirect” kit for ISH. Nanogold was developed by Dr. James F. Hainfeld [20–23, 45] and is available from and patented by Nanoprobes, Inc. (Yaphank, NY; http://www.nanoprobes.com/index.html). 6.2.1.1
Materials and Reagents
DAKO-Pen: DakoCytomation, Cat. No. S-2002 (Glostrup, Denmark and Carpinteria, CA) (http://www.dakocytomation.com). 2.5% H2O2 solution in absolute methanol. Double-distilled (ultrapure) water. Proteinase-K: from BoehringerMannheim/Roche Diagnostics Corporation (Indianapolis, IN), or from DakoCytomation; Cat. No. S3004. 110
Deionized formamide (e.g., from SigmaAldrich, Steinheim, Germany). Dextran sulfate, 20% (e.g., from Chemicon, http://www.chemicon.com). Streptavidin-biotin-peroxidase reagent (e.g., from HRP Streptavidin-Biotin Duet Kit, Cat. No. K0492, DakoCytomation). Lugol’s iodine solution: Code No. 109261, Merck, West Point, PA. 2.5% Aqueous sodium thiosulfate solution (store at room temperature). “Blocking solution”: 4X SSC containing 5% casein sodium salt (Cat. No. C8654, Sigma), or from the DakoCytomation CSA-kits (e.g., the “GenPoint” kit, Cat. No. K0620). Tween-20 (e.g., Cat. No. 1332 465, Boehringer Mannheim/Roche). Biotinylated Tyramides: either from the “TSA Biotin System” (e.g., Cat. No. NEL700A001T, Perkin-Elmer Life and Analytical Sciences, http://las. perkinelmer.com/catalog/Product.aspx ?ProductID=NEL700A001KT, or contained in the “GenPoint” kit, Cat. No. K0620, DakoCytomation (http://www.dakocytomation.us). The BT reagent in the DakoCytomation GenPoint kit is ready to use. For the NEN kit, a stock solution of BT is prepared by adding 100 ml ethanol to the lyophilized reagent and is diluted 1/50 to 1/100 with the supplied amplification diluent mixed with distilled water at 1:1, as described in the kit. According to the guidelines supplied, the working solution should contain 1 or 0.5 mg BT per milliliter diluent, consisting of 0.2 M Tris-HCl, 10 mM imidazole, pH 8.8 and 0.01% H2O2. 20% DMSO (dimethyl sulfoxide; e.g., http://www.dmso.org/). Phosphate-buffered saline (PBS), pH 7.6: For a 10× PBS stock solution
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Toward Molecular Sensitivity (0.1 mM PBS), dissolve 11.36 g Na2HPO4, 2.72 g KH2PO4, and 87.0 g NaCl in 1 L distilled water. Before use, dilute 1:10 in high quality distilled and preferably also deionized water. PBS-gelatin: PBS containing 0.1% fish gelatin (Teleostean gelatin from coldwater fish skin; Code No. G-7765; Sigma-Aldrich, Steinheim, Germany). 1% Bovine serum albumin (BSA), in PBS. Streptavidin-Nanogold: Cat. No. 2016, Nanoprobes (http://www.nanoprobes. com/index.html). SSC-buffer: A 20X concentrated SSC aqueous stock solution is prepared as follows: NaCl 175.32 g/L (3 M), Na3-citrate·2 H2O 88, 23 g/L (0, 3 M). Heating block (95 ± 3°C); for example, Enzo, New York, NY, (http://www.enzobio.com). Plastic forceps: avoid contamination with metal shortly before or during the AMG enhancement process. Nuclear Fast Red counterstain (“Kernechtrot”): Code No. 115939, Merck. Warm up to about 50°C in a microwave oven before use. This solution is reusable. Mounting medium: DPX (Electron Microscopy Sciences (EMS), Fort Washington, PA, or Permount (e.g., Biomeda, Cat. No. M17, http://biomeda.com/site/M17.pdf ) Silver or gold AMG enhancement solutions (see Protocols in Section 6.2.3.1 or 6.2.3.2). 6.2.1.2 1.
2. 3. 4. 5.
6. 7. 8.
9. 10. 11. 12. 13.
14. 15. 16.
Staining Procedure
Deparaffinize formaldehyde-fixed sections in fresh xylene (2 × 15 min each).
17.
Rinse in absolute ethanol (2 × 5 min each). Treat with 3% H2O2 in methanol at room temperature for 30 min. Rinse in double-distilled (ultrapure) water for 10 sec and then in PBS for 3 min. Incubate sections with 0.1 mg/ml proteinase-K in PBS at 37×C for about 8 min (optimal duration should be tested). Wash in two changes of PBS, 3 min each, then ultrapure water for 10 sec. Dehydrate with graded alcohols (50%, 70%, absolute ethanol) for 5 min each and air-dry the sections. Prehybridize with 1:1 mixture of deionized formamide and 20% dextran sulfate in 2× SSC at 50°C for 5 min. Carefully shake off the excess prehybridization block. Add one drop of biotinylated DNA probe on the section and cover with a small coverslip. Avoid air bubbles. Heat sections on heating block at 92 to 94°C for 10 min to denature DNA. Incubate in a moist chamber at 37°C overnight (or for at least 2 hr). Post-hybridization washes (5 min each): two changes of 2X SSC (first wash to remove coverslips), 0.5X SSC, 0.2X SSC, and then distilled water. Put slides into Lugol’s iodine solution for 5 min. Wash in tap water and then doubledistilled water. Put into 2.5% sodium thiosulfate for a few seconds until sections are colorless. Then wash in distilled water for 2 min. Drain off section, wipe area around section dry, and surround it with a PAP-pen. 111
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Molecular Morphology in Human Tissues: Techniques and Applications 18. Incubate with blocking solution at 37°C for 30 min. 19. Briefly wash in 4X SSC containing 0.05% Tween 20, 2 min. 20. Incubate with streptavidin-biotinperoxidase complex (e.g., from DakoCytomation “duet kit”) at room temperature for 30 min. The complex is dissolved in the above blocking solution at a concentration of 1:200. 21. Wash in three changes of 4X SSC containing 0.05% Tween 20 for 2 min each, followed by two changes of PBS for 2 min each. 22. Incubate the sections with biotinylated tyramide (BT) at room temperature for exactly 10 min (with BT from Renaissance TSA-Indirect ISH Kit, Perkin-Elmer) or 15 min (with BT from “GenPoint” kit, DakoCytomation). 23. Wash in four changes of PBS containing 0.05% Tween 20 and 20% DMSO (dimethyl sulfoxide) at room temperature for 3 min each. 24. Immerse in PBS-gelatin (PBS containing 0.1% fish-gelatin) for 5 min. 25. Incubate the sections with streptavidin-Nanogold (Nanoprobes) diluted 1:750 in PBS containing 1% BSA at room temperature for 60 min. 26. Wash in PBS, 2 × 5 min each. 27. Wash in PBS-gelatin for 5 min. 28. Repeatedly wash in ultrapure water (EM grade). Proceed with Section 6.2.3 for silveror gold-ion-based AMG enhancement of the Nanogold label. 6.2.1.3 Results, Applications, and Technical Hints Under optimum circumstances (adequate fixation and preparation of the tissue, adequate sectioning and processing, well-performing hybridization probe), the technique described here gives distinct jet112
black staining of the DNA sequence to be detected, even if only single copies of the sequence in question are present (Figure 6.1). In our experience, paraffin/paraplast tissue blocks from routine pathology files of up to 20 years old often worked successfully, partly even more successful than detection with PCR on extracted DNA was performed in parallel [28, 29]. For DNA, biotinylated genomic probes were used most successfully, whereas oligoprobes have not yet been tested conclusively by us. For RNA detection, biotinylated riboprobes gave excelent results (e.g., Tubbs in [27]). Rigorous specificity testing is necessary in any in situ hybridization reaction — which becomes especially pertinent when using such highly detection-efficient techniques. Controls should include omission of the probe in the hybridization mixture, as well as the parallel use of known-positive and -negative tissue sections or cytological material. When testing a new system, it is mandatory to separately check and optimize each step of the entire procedure, including post-hybridization stringency washes, as well as various steps in the detection protocol [27]. For streptavidin-Nanogold, staining intensity can be adjusted by simple dilution experiments (“titration”) and/or varying conditions of the AMG procedure applied. It was observed that less-experienced lab personnel sometimes produced staining with high background, even when the rest of the procedure was well adjusted. The degree of staining intensity is related to the duration and temperature of enhancement; it can be represented as a linear histogram reflected by the size of the growing gold-silver or gold-gold particles [27, 44]. For the detection of RNA, it is important to bear in mind a number of precautions that apply to every kind of
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Figure 6.1 Human papillomavirus (HPV) 16/18 detected in dedifferentiated cervical squamous cell carcinoma using the protocol described in Section 6.2.1 in combination with GoldEnhance® development. In parallel, sections of SiHa cells were stained, known to contain only one or two copies of HPV 16 per cell. Staining appearance and intensity was comparable in both preparations; therefore, we can assume that the carcinoma section also contained only one copy of HPV16/18 in each of the tumor cells, visible here as a black spot seen within the nuclei. 4-µm thick, formalin-fixed paraffin section, mounted on a silanized glass slide, counterstained with Nuclear Fast Red; 100× objective.
RNA hybridization: RNAases are ubiquitous and both the RNA target and the riboprobe can readily be degraded. The investigator must carefully minimize this risk by wearing gloves during the experiment and using autoclaved glassware. 6.2.2 The GOLDFISH Procedure (Gold-Facilitated Autometallographic In Situ Hybridization) The GOLDFISH procedure, gold-facilitated autometallographic in situ hybridization, was developed specifically for the detection of HER2 gene amplification in invasive mammary carcinoma, but its principles can at least in theory apply to any gene for profiling amplification or deletion. The goal of the GOLDFISH procedure was a radial signal growth via a combination of tyramide signal amplification and AMG. Ultimately, the method was
designed to obviate the need for signal enumeration, as is commonly performed for fluorescence in situ hybridization, and produce signals of such comparatively large size as to permit interpretation with conventional bright-field microscopy. Specifically, the use of oil immersion microscopy was considered an impediment to routine interpretation of molecular morphology genotyping preparations, and the large signal size was specifically designed to avoid the use of oil immersion microscopy. The GOLDFISH procedure leverages the remarkable chemical field effects of tyramide signal amplification, whereby the deposition of very large amounts of a hapten or ligand linked to tyramide is achieved. Further signal amplification is achieved by radial expansion through deposition of metallic gold upon the nidus of the same metal as a constituent of Nanogold-labeled streptavidin (Nanoprobes, Yaphank, NY). 113
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Molecular Morphology in Human Tissues: Techniques and Applications 6.2.2.1
Materials and Reagents
The procedure has been optimized for paraffin sections on electrostatically charged slides (Superfrost; Fisher; Cat. No. M6146-PLUS) of formalinfixed breast cancer tissue. Other fixatives yield inconsistent results. Xylene (Allegiance, McGaw, IL; Cat. No. C4330) Dehydrant (Richard-Allan Scientific, Richland, MI; Cat. No. 6201) Distilled water Dako Target Retrieval Solution (DakoCytomation, Carpinteria, CA; Cat. No. S1700) Proteinase K (DakoCytomation; Cat. No.S3004) Tris buffer 50 mM from Trizma HCl (Sigma, St. Louis, MO; Cat. No. T2444) H2O2 30% (Sigma; Cat. No. H-0904) Biotin-labeled HER2 probe (Ventana Medical Systems International, Tuscon, AZ; Cat. No. 780-2840) SSC 20X (Sigma; Cat. No. S-6639) 0.1% Tween 20 (Sigma; Cat. No. P9416) HRP – Streptavidin (DakoCytomation; GenPoint Kit; Cat. No. K0620) TSA-Biotin (DakoCytomation; GenPoint Kit; Cat. No. K0620) Lugol’s iodine (Merck, West Point, PA; Cat. No. 109261) Sodium thiosulfate 2.5% in distilled water (Mallinckrodt, Hazelwood, MO. Cat. No. 7763) PBS 10X (Gibco/Invitrogen, Grand Island, MI; Cat. No. 70011-044) PBS – Gelatin; PBS containing 0.1% fish gelatin (Sigma; Cat. No. G7765) Streptavidin Nanogold (Nanoprobes, Yaphank, NY; Cat. No. 2016) GoldEnhance LM/Blot (Nanoprobes; Cat. No. 2112) 114
Neutral Fast Red counterstain (Newcomer, Middleton, WI, Cat. No. 1255B) Cytoseal/60 (Richard-Allen, Richland, MI; Cat. No. 8310-4) 6.2.2.2 1.
2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Staining Procedure
Prepare 4- to 6-µm-thick unstained sections on electrostatically charged slides, and dry overnight at room temperature. Deparaffinize and rehydrate sections in xylene and graded alcohols. Soak sections in distilled water for 5 min at room temperature. Incubate sections with Target Retrieval Solution for 40 min at 95°C, then cool down at room temperature for 20 min. Rinse sections in distilled water, 5 min each, several changes. Incubate sections with proteinase-K, 1:5000 in 50 mM Tris for 4 min at room temperature. Rinse sections for 5 min in distilled water, several changes. Quench endogenous peroxidase activity with 3% H2O2 in methanol for 20 min at room temperature. Rinse sections in distilled water for 20 min at room temperature. Dehydrate sections in graded alcohols. Apply 10 µl of the biotinylated HER2 probe; cover with a glass coverslip and seal with rubber cement. Co-denature probe and target tissue for 6 min at 90°C on a heating block. Hybridize at 37°C overnight in humidified chamber. Remove coverslip by soaking in 2× SSC for 5 min. Perform stringent wash, 0.5× SSC for 5 min at 72°C.
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Toward Molecular Sensitivity 16. Wash in 0.1% Tween 20 in 1× PBS for 3 min at room temperature. 17. Apply Strepavidin-HRP, 100 µl, for 15 min at room temperature. 18. Wash sections in 0.1% Tween 20 in 1× PBS for 5 min, three separate washes, at room temperature. 19. Apply prediluted TSA biotin for 10 min at room temperature. 20. Wash sections in 0.1% Tween 20 and 1× PBS for 5 min, three washes over 5 min at room temperature. 21. Apply Lugol’s iodine by immersing slides for 5 min at room temperature. 22. Wash in three rinses of double-distilled water. 23. Immerse slides for a few seconds in 2.5% sodium thiosulfate until the tissue color clears. 24. Wash in double-distilled water for three to five rinses over 7 min total. 25. Apply 0.1% fish gelatin in PBS pH 7.6 and immerse for 5 min. 26. Apply Strepavidin-Nanogold prediluted to 1:400 in PBS pH 7.6 with 1% bovine serum albumin, 50 µl per application, and incubate for 30 min at room temperature. 27. Wash sections in PBS pH 7.6, two washes at 5 min each. 28. Immerse slides in fish gelatin for 5 min at room temperature. 29. Rinse in double- distilled water for 10 min using several changes. 30. Add parts A and B of Nanoprobes GoldEnhance kit to the sections; after 10 min, add reagents C and D from the GoldEnhance kit, and develop the reaction for 4 min. Stop GoldEnhance reaction by addition of 500 µm of 2.5% sodium thiosulfate to the slide, and rinse well in distilled water. (Note: In some cases, sodium thiosulfate might remove the staining — which was especially observed in combination with tyramides. Therefore, stopping
the development process might also be achieved by simply washing sections thoroughly in distilled water.) 31. Counterstain sections with Nuclear Fast Red for 8 min. 32. Dehydrate sections in graded alcohols in xylene and coverslip. 6.2.2.3 Results, Applications, and Technical Hints For clinical use of the GOLDFISH procedures, we have found that the assembly of a study set comprising a brief tutorial for all the pathologists interpreting the GOLDFISH slides is invaluable. The study set consists of three positive and three negative cases, along with specific instructions regarding the sequence of microscope objectives to be used and the capability of interpreting the slides without oil immersion objectives. Nonamplified cases demonstrate one or two small dense black signals in about 30 to 50% of nuclei; not every nucleus is positive because the gene target is not present in every nucleus of a 4-µm paraffin section that contains both intact and truncated nuclei. Amplified cases show obvious massive confluent black dense granules that are most commonly centronuclear (Figure 6.2). Each reviewer should have both a presection H&E and the GOLDFISH preparation. Interpretation begins with a 10× objective and progresses to higher magnifications, but only in very rare circumstances are oil immersion objectives required. 6.2.3 Autometallography: General Guidelines AMG is the general term used for detection and enhancing methods of certain heavy and noble metals, as introduced by Danscher [18, 35–39]. AMG relies on the 115
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Figure 6.2 Photomicrograph demonstrating Gold-Facilitated Autometallographic In Situ Hybridization (GOLDFISH), using the protocol in Section 6.2.2. Massive confluent centronuclear deposition of the autometallographic product of GoldEnhance is identified in tumor cells characteristic of amplification of the HER2 gene. Specimens were counterstained with neutral Fast Red and examined with conventional light microscopy. (Original magnification 400×.)
controlled deposition of metallic silver or of metallic gold onto the surface of catalytic metals, such as colloidal gold or clustered gold (Nanogold). The method has been reviewed in detail in various chapters of the book Gold and Silver Staining: Techniques in Molecular Morphology, also published by CRC Press in the series Advances in Pathology, Microscopy & Molecular Morphology (see also Chapter 4 of the present book). Precipitation of gold or silver metal onto gold particles in effect increases the size of the originally very tiny (1.2-nm gold core diameter) Nanogold particles to sizes readily visible in the LM. As soon as the density of gold particles at the reaction site is high enough, conglomeration takes place. It is crucial to use very clean water to obtain optimal staining results. For all washes shortly before and during the AMG enhancement process, it is highly recommended to use glass-distilled water. Even minimal impurities (e.g., chloride) will induce a precipitate and render the 116
enhancement solutions useless. For the same reason, it is highly important to use very clean glassware and to avoid contact between metallic objects (use plastic forceps) and the silver or gold solutions. Speed and outcome of AMG reactions depend on various factors. It has turned out that neutral pH conditions in some cases tend to yield argyrophilic or argentaffin-type reactions [48]; therefore, laboratory-made AMG developers should always use low pH buffers, as is the case in the silver acetate AMG method described below. Commercial developers sometimes do not follow this well-documented rule; therefore, their use should be exercised with great care. The GoldEnhance developer described below is a new and very convenient method of gold enhancement, and the results are of extraordinarily high distinctness and beauty. In our hands, no unspecific (argyrophilic and/or argentaffin-type) reactions have been observed under these ameliorated and convenient
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Toward Molecular Sensitivity conditions.The size of the gold-silver or gold-gold particles increases with time. Usually, when observed with the “naked eye,” one cannot see a color change for some minutes. The, the sites of reaction start to change color, and in most cases, some kind of brownish or grayish color starts to come up. As soon as a relevant signal level is observed with the naked eye, it is suggested to check one of the sections under the LM to optimize staining strength. As soon as an ideal result appears to be reached, the reaction on this slide, or on all slides stained in parallel, should be stopped by rinsing the sections or cells in double-distilled water. Some earlier protocols suggested stopping the AMG reaction immediately via a quick and short dip in 2.5% sodium thiosulfate, followed by washing in distilled water. We do not recommend this anymore, as we have observed that this step sometimes introduces a complete removal of the black gold-silver or gold-gold stain. Dipping or rinsing in double-distilled water carried out instead, as described above, stops the enhancement process a bit slower; however, this does not affect the final outcome. The “water way” also brings an additional advantage: when the reaction observed in the LM is not sufficiently black, the slides can be further developed in AMG solution, thereby enabling the experimenter to achieve optimized staining results. After AMG amplification, sections can be counterstained with hematoxylin and eosin and/or Nuclear Fast Red, dehydrated and mounted in Permount or in DPX. Other coverslipping glues should be used with reservation only. Eukitt, for example, has a tendency to remove the AMG stain after minutes, hours, days, or weeks. An acceptable alternative might be Canada balm; we do not use this anymore, as it is a very “sticky” procedure.
6.2.3.1 Silver Acetate Autometallography Silver acetate AMG allows the reliable enhancement colloidal gold or of clustered gold (Nanogold) under microscopic control and therefore can be used to yield optimal signal-to-noise ratio [18]. Although the principle of silver acetate AMG is being used in a number of commercially available “light-insensitive silver enhancement kits,” it is most often more economical to perform it following the original protocol. Using a non-neutral pH in the citrate buffer system better ensures that no unwanted nonspecific reactions would occur. Procedure: Solutions A and B should be freshly prepared for every run. Solution A: dissolve 80 mg silver acetate (Code No. 85140; Fluka, Buchs, Switzerland) in 40 ml of glass double-distilled water. Continuous stirring within about 15 min can dissolve silver acetate crystals easily. Citrate buffer: dissolve 23.5 g trisodium citrate dihydrate and 25.5 g citric acid monohydrate in 850 ml deionized or distilled water. This buffer can be kept at 4°C for at least 2 to 3 weeks. Before use, adjust to pH 3.8 with citric acid solution. Solution B: dissolve 200 mg hydroquinone in 40 ml citrate buffer. Enhancement solution: just before use, mix solution A with solution B. Silver amplification: place the slides vertically in a glass container (preferably with about 80-ml volume and up to 19 slides; Schiefferdecker type) and cover them with the mixture of solutions A and B. Staining intensity can be checked in the LM during the amplification process, which usually takes about 5 to 20 min, depending on probe concentration, the quality and 117
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Molecular Morphology in Human Tissues: Techniques and Applications concentration of biotinylated tyramides, the overall incubation conditions, and the amount of accessible nucleic acid sequence in question. After stopping the enhancement process by immediate and thorough rinsing in double-distilled water, slides can be examined more carefully in a LM. If staining intensity is too low, wash slides one more time in double-distilled water and develop further in enhancement solution. Counterstaining can be applied following optimization of the specific staining result and additional washing in distilled water for at least 3 min. Nuclear Fast Red (“Kernechtrot”), and/or hematoxilin, and/or eosin are good choices. If only a few specific reaction sites are present, only red counterstains (e.g., Nuclear Fast Red followed by eosin, but without hematoxilin) can improve screening in the search for stained hybridization sites. For good overall counterstaining, a combination of the three stains, in which the hematoxilin stain is carried out in a light version, appears adequate. 6.2.3.2 GoldEnhance Autometallography GoldEnhance autometallography represents an advance in both the performance of metallographic ISH and immunohistochemistry, and also significant gains in ease of use for laboratory personnel. The technique depends on radial growth of signal by deposition of metallic gold onto the nidus of the Nanogold particle, and yields excellent preparations of high resolution visible by conventional bright-field microscopy. Procedure: The GoldEnhance Kit (Nanoprobes, Inc., Yaphank, NY) is available as a very user-friendly kit containing four reagents: that is Reagents A, B, C, and 118
D. No special preparation of the component parts is required. 1.
2.
3.
4. 5.
Combine equal volumes of Reagents A and Reagent B, mix well, dispense onto slide, and incubate for 10 min at room temperature. Combine Reagents C and D from GoldEnhance Kit, mix well, dispense onto slide, and develop for 4 min at room temperature. Stop the GoldEnhance reaction with the addition of 500 µl of 2.5% sodium thiosulfate applied to the slide and rinse well in distilled water. (Note: Sometimes it is necessary to avoid the use of sodium thiosulfate and replace it by thorough rinsing in distilled water, as is the case in tyramide-related techniques. Sodium thiosulfate has, in rare cases, removed or reduced staining intensity.) Counterstain slides with Fast Red for 8 min. Dehydrate sections in graded alcohols in xylene and coverslip.
The reaction time of 4 min works very well for applications employing HER2 probes. In the early stages of optimizing the experimental conditions, it may be helpful to examine other incubation times, as the recommended duration of 4 min may not be optimal for all experimental conditions. 6.3 DISCUSSION The outcome of super-sensitive ISH depends on numerous factors, such as the basic hybridization conditions, quality of the nucleic acid probe applied, the concentrations, temperatures and durations of the various incubation steps, stringent washings, etc. For this, the reader is referred to a variety of specialized literature (see, for example, [49]). As “single copy sensitivity”
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Toward Molecular Sensitivity is the target, it is crucial to optimize each of these factors. Tyramide signal amplification/catalyzed reporter deposition (TSA or CARD) is an extraordinarily sensitive technique with enormous potential for signal amplification in both IHC and ISH systems. Its inherent remarkable sensitivity is also accompanied by the potential for augmented artifacts, and it is the molecular morphologist’s challenge to balance these two extremes. The CARD reaction fundamentally differs from most reagent component interactions in IHC and ISH. Whereas the reaction between biotin and avidin, and between antigen and antibody, are precisely linked by specific binding properties, and derive their analytical utility from this very precise binding interaction, the TSA and CARD techniques actually depend on a “chemical field effect” that must also be carefully addressed and controlled when optimizing experimental conditions. The use of the hapten/ligand attached to the tyramide is an important consideration in optimizing experimental conditions. Commonly employed ligands include biotin, fluorescein isothiocyanate (FITC), and dinitrophenyl (DNP). Each of these ligands may offer certain advantages and be associated with limitations more related to the availability of high-quality secondary antibodies having specificity for the DNP, biotin, or FITC; and of course, streptavidin or avidin-enzyme or fluorescent conjugates also vary widely in performance characteristics and availability. If a biotin tyramide system does not function properly, it may be prudent to simply switch to another ligand-tyramide conjugate. A very significant variable in achieving optimal performance in a CARD system depends on the quality of the streptavidin peroxidase used for the oxidation of the
tyramide-ligand conjugate. It is very important that this particular reagent, streptavidin peroxidase used to catalyze the tyramide-ligand conjugate is of the utmost purity and enzyme activity, to achieve success. In this regard, the “primary” streptavidin peroxidase in the DakoCytomation Genpoint kit is especially well suited to this purpose. A thorough washing with appropriate buffers is a commonly overlooked but critical component in the success of the CARD procedure. Following catalysis of the tyramide-ligand conjugate, there must be vigorous removal of any free noncatalyzed tyramide in the solution overlying the section, because this residual ligand will preferentially react with any peroxidasecontaining reagents used subsequently in the detection. This is a very common source for both insufficient signal detection and excessive nonspecific background staining artifacts. In our experience, it is not simply enough to vigorously and repeatedly wash the sections with buffer; rather, heating the section to 60°C with at least three changes of the washing buffer are critical in achieving success. 6.4 OUTLOOK The use of autometallography in conjunction with the CARD reaction leverages the combined enhanced sensitivity of both approaches to produce outstanding preparations visible by conventional light microscopy (LM). Future applications of the technique are legion, and include (1) combinations of immunohistochemistry with in situ hybridization to simultaneously visualize gene amplification and encoded protein expression; (2) the combination of protein-based studies with mRNA in situ hybridization using CARD and autometallography; and (3) simultaneous evaluation 119
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Molecular Morphology in Human Tissues: Techniques and Applications of the state of amplification at the genomic level via DNA in situ hybridization, combined with in situ hybridization for mRNA, yielding important information regarding the status of gene expression, particularly for those genes that are only identified as expressed sequence tags (ESTs) in cDNA expression array experiments. The potential for three-color systems by which the DNA, mRNA, and encoded protein can be concommitantly assessed on a single histologic slide is clearly possible with this technology, and no doubt will be developed in the future.
2.
3.
4.
5.
Acknowledgments
The procedures described were developed in joint collaboration and friendship with a number of distinguished scientists, listed here in alphabetical order: Annie L.M. Cheung (Hong Kong, PR China), Gorm Danscher (Aarhus, Denmark), Anton-Helmut Graf (Salzburg, Austria), Lars Grimelius (Uppsala, Sweden), Thomas Grogan (Tucson, AZ), Jiang Gu (Brooklyn, NY, and Beijing, PR China), James Hainfeld (Brooklyn, NY), Cornelia Hauser-Kronberger (Salzburg, Austria), David Hicks (Cleveland, OH), James Pettay (Cleveland, OH), Julia M. Polak (London, U.K.), Rick Powell (Yaphank, NY), the late David R. Springall (London, U.K.), Richard Huici Su (San Francisco, CA), and Ingeborg Zehbe (Mainz, Germany). We would like to express our deep thanks to all of them.
6.
7.
8.
9.
10.
11.
References 12. 1.
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Cremers, A.F. et al., Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy, Histochemistry, 86, 609, 1987. Giaid, A. et al., Non-isotopic RNA probes. Comparison between different labels and detection systems, Histochemistry, 93, 191, 1989. Scippo, M.L. et al., A non-radioactive method to detect RNA or DNA using an oligonucleotide probe with bromodeoxyuridine free ends, a monoclonal antibody against bromodeoxyuridine and immunogold silver staining, Arch. Int. Physiol. Biochim., 97, 279, 1989. Varndell, I.M. et al., Visualisation of messenger RNA directing peptide synthesis by in situ hybridisation using a novel single-stranded cDNA probe. Potential for the investigation of gene expression and endocrine cell activity, Histochemistry, 81, 597, 1984. Coggi, G., Dell’Orto, P., and Viale, G., Avidinbiotin methods, in Immunocytochemistry — Modern Methods and Applications, Polak, J.M. and Van Noorden, S., Eds., Wright, Bristol, U.K., 1986, 54–70. Hacker, G.W., High-performance Nanogold-silver in situ hybridisation, Eur. J. Histochem., 42, 111, 1988. Liesi, P. et al., Specific detection of neuronal cell bodies: in situ hybridization with a biotinlabeled neurofilament cDNA probe, J. Histochem. Cytochem., 34, 923, 1986. Hacker, G.W. et al., Application of silver acetate autometallography and gold-silver staining methods for in situ DNA hybridization, Chin. Med. J. (Engl.), 106, 83, 1993. Zehbe, I., Rylander, E., Strand, A., and Wilander, E., Use of Probemix and OmniProbe biotinylated cDNA probes for detecting HPV infection in biopsy specimens from the genital tract, J. Clin. Pathol., 46, 437, 1993. Tanner, M. et al., Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples, Am. J. Pathol., 157, 1467, 2000. Hacker, G.W. and Gu, J., Gold and Silver Staining: Techniques in Molecular Morphology, CRC Press, Boca Raton, FL, 2002. Teo, C.G. and Griffin, B.E., Visualization of single copies of the Epstein-Barr virus genome by in situ hybridization, Anal. Biochem., 186, 78, 1990.
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Toward Molecular Sensitivity 14. Hacker, G.W. et al., High-performance Nanogold in situ hybridization and in situ PCR, Cell Vision, 3, 209, 1996. 15. Bagasra, O., Polymerase chain reaction in situ, Amplifications, March, 20, 1990. 16. Haase, A.T., Retzel, E.F., and Staskus, K.A., Amplification and detection of lentiviral DNA inside cells, Proc. Natl. Acad. Sci. U.S.A., 87, 4971, 1990. 17. Zehbe, I. et al., Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction (in situ PCR) combined with immunoperoxidase and immunogold-silver staining (IGSS) techniques, Anticancer Res., 12, 2165, 1992. 18. Hacker, G.W. et al., Silver acetate autometallography: an alternative enhancement technique for immunogold-silver staining (IGSS) and silver amplification of gold, silver, mercury, and zinc in tissues, J. Histotechnol., 11, 213, 1988. 19. Hacker, G.W. and Danscher, G., Recent advances in immunogold-silver stainingautometallography, Cell Vision, 1, 102, 1994. 20. Hainfeld, J.F., A small gold-conjugated antibody label: improved resolution for electron microscopy, Science, 236, 450, 1987. 21. Hainfeld, J.F., Gold cluster-labelled antibodies, Nature, 333, 281, 1988. 22. Hainfeld, J.F. and Furuya, F.R., A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177, 1992. 23. Hainfeld, J.F., Labeling with nanogold and undecagold: techniques and results, Scanning Microsc. Suppl., 10, 309, and discussion 322, 1996. 24. Hacker, G.W. et al., Electron microscopical autometallography: immunogold-silver staining (IGSS) and heavy-metal histochemistry, Methods, 10, 257, 1996. 25. Zehbe, I. et al., Self-sustained sequence replication-based amplification (3SR) for the in situ detection of mRNA in cultured cells, Cell Vision, 1, 20, 1994. 26. Zehbe, I. et al., Sensitive in situ hybridization with catalyzed reporter deposition, streptavidinNanogold, and silver acetate autometallography: detection of single-copy human papillomavirus, Am. J. Pathol., 150, 1553, 1997. 27. Hacker, G.W., High performance Nanogold-silver in situ hybridisation, Eur. J. Histochem. 42, 111, 1998.
28. Cheung, A.L. et al., Detection of human papillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization, Mod. Pathol., 12, 689, 1999. 29. Graf, A.H. et al., Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma, Appl. Immunohistochem. Mol. Morphol., 8, 300, 2000. 30. Bobrow, M.N. et al., Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays, J. Immunol. Meth., 125, 279, 1989. 31. Bobrow, M.N., Shaughnessy, K.J., and Litt, G.J., Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays, J. Immunol. Meth., 137, 103, 1991. 32. van Gijlswijk, R.P. et al., Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization, J. Histochem. Cytochem., 45, 375, 1997. 33. Tbakhi, A. et al., Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma, Am. J. Pathol., 152, 35, 1998. 34. Hacker, G.W. et al., In situ localization of DNA and RNA sequences: super-sensitive in situ hybridization using streptavidin-Nanogold-silver staining: minireview, protocols, and possible applications, Cell Vision, 4, 54, 1997. 35. Danscher, G., Light and electron microscopic localization of silver in biological tissue, Histochemistry, 71, 177, 1981. 36. Danscher, G., Localization of gold in biological tissue. A photochemical method for light and electronmicroscopy, Histochemistry, 71, 81, 1981. 37. Danscher, G., Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electronmicroscopy, Histochemistry, 71, 1, 1981. 38. Danscher, G. and Norgaard, J.O., Light microscopic visualization of colloidal gold on resinembedded tissue, J. Histochem. Cytochem., 31, 1394, 1983. 39. Danscher, G., Autometallography. A new technique for light and electron microscopic visualization of metals in biological tissues (gold, silver, metal sulphides and metal selenides), Histochemistry, 81, 331, 1984.
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Molecular Morphology in Human Tissues: Techniques and Applications 40. Holgate, C.S. et al., Surface membrane staining of immunoglobulins in paraffin sections of nonHodgkin’s lymphomas using immunogold-silver staining technique, J. Clin. Pathol., 36, 742, 1983. 41. Holgate, C.S. et al., Immunogold-silver staining: new method of immunostaining with enhanced sensitivity, J. Histochem. Cytochem., 31, 938, 1983. 42. Hacker, G.W. et al., The immunogold-silver staining method. A powerful tool in histopathology, Virchows Arch. A Pathol. Anat. Histopathol., 406, 449, 1985. 43. Springall, D.R. et al., The potential of the immunogold-silver staining method for paraffin sections, Histochemistry, 81, 603, 1984. 44. Lackie, P.M. et al., Investigation of immunogold-silver staining by electron microscopy, Histochemistry, 83, 545, 1985. 45. Hainfeld, J.F. and Powell, R.D., New frontiers in gold labeling, J. Histochem. Cytochem., 48, 471, 2000.
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46. Tubbs, R. et al., Interobserver interpretative reproducibility of GOLDFISH, a first generation gold-facilitated autometallographic bright field in situ hybridization assay for HER-2/neu amplification in invasive mammary carcinoma, Am. J. Surg. Pathol., 26, 908, 2002. 47. Tubbs, R. et al., Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of Her-2/neu gene amplification, Am. J. Pathol., 160, 1589, 2002. 48. Hacker, G.W. et al., Silver staining techniques, with special reference to the use of different silver salts in light- and electron microscopical immunogold-silver staining, in Immunogold-Silver Staining: Methods and Applications, Hayat, M.A., Ed., CRC Press, Boca Raton, FL, 1995, 20–45. 49. Polak, J.M., Wharton, J., and McGee, J. O’D., Eds., In Situ Hybridization: Principles and Practice, 2nd ed., Oxford University Press, Oxford, U.K., 1999.
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Array-Based Comparative Genomic Hybridization as a Tool for Survey of Genomic Alterations in Human Neoplasms Dina Kandil, Marek Skacel, James D. Pettay, and Raymond R. Tubbs
7.1 INTRODUCTION Microarrays have been exploited most extensively for gene expression studies, but other applications have been recently developed, including the use of arrays for the detection of gains and losses of genomic DNA. Fluctuations in DNA sequence copy number with concomitant microscopic or cryptic chromosomal aberrations are becoming increasingly correlated with phenotypic abnormalities [1]. Gene amplification and deletion are common alterations occurring in cancer cells and, like other structural changes, are associated with genomic instability and contribute to the process of carcinogenesis. The development of most human neoplasms follows a defined series of histopathological stages, a process that involves multiple genetic changes such as translocations, deletions, duplications, and alterations in chromosomal copy number changes. Importantly, the DNA amplification or losses frequently involve oncogenes and tumor suppression genes that play an important role in the cell cycle control and their alteration affects cancer growth. 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
It is now possible to screen tumors for genomic changes, as an alternative to the examination of the transcriptional activity of the tumor cells. Compared to mRNA expression, the genomic changes are less variable and are less likely to be subject to transient changes in the tissue environment [2]. This reduced complexity greatly simplifies the identification of causal genetic changes in individual tumors. Also, the dynamic range of the genomic changes is more manageable (the locus-tolocus variation of DNA is small, including either allele loss or locus amplification), while messenger RNA levels present in cells typically vary over several orders of magnitude. The knowledge of genetic changes common to specific tumor types may facilitate the development of markers for early detection, diagnosis, and monitoring during clinical intervention. Furthermore, the identification of these genomic alterations may define the causal molecular changes leading to more targeted therapeutic approaches to disease. The best illustration of the power of this approach is the identification of the HER2 gene amplification in breast cancer, which
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Molecular Morphology in Human Tissues: Techniques and Applications has not only provided a prognostic criterion but also led to the development of targeted therapy [3]. Classical cytogenetics has been used for decades to karyotype cells but is limited due to the need to culture the cells in order to produce metaphase chromosomes, and requires expertise in interpreting the chromosomal spreads. In addition, cytogenetics only provides a crude analysis of the chromosome number and has limited sensitivity in identifying deletions and amplifications. The introduction of comparative genomic hybridization (CGH) to metaphase chromosomes (M-CGH) revolutionized clinical cytogenetics by permitting a genome-wide analysis of cancer specimens with chromosomal aberrations that were either too many or too complex to be fully characterized by routine cytogenetics [4]. Moreover, because CGH requires only genomic DNA from the sample, it permits the analysis of specimens from which chromosomal preparations are impossible to obtain due to poor cell growth. Using this technique, the genomic DNA from the tumor is labeled with a fluorescent dye in one color while a normal reference DNA sample is labeled in a different color. The labeled samples are then co-hybridized to normal metaphase chromosome spreads. Chromosomal imbalances across the genome in the tumor DNA are then quantified and positionally defined by analyzing the ratio of fluorescence of the two different colors along the target metaphase chromosomes [5]. However, the resolution of CGH applied to the metaphase spreads is limited by cytogenetic resolution of approximately 5 Mb, and a considerable cytogenetics expertise is required to accomplish such analysis. Therefore, M-CGH has never 124
become a widely utilized technique, and remains limited to specialist research applications. The inherently low resolution associated with metaphase chromosomes banding, along with the labor intensiveness of this procedure, make M-CGH largely incapable of accomplishing genome-wide screens for chromosomal aberrations that are less than 5 Mb in size, a limitation that makes this approach unsuitable for a high-throughput study of human neoplasms. Recently, the advent of bacterial artificial chromosome (BAC) array technology has redrawn the attention to the applicability of CGH to the study of genomic alterations in human disease. BACs are large-insert DNA clones that have been cytogenetically and physically mapped to the human genome. Currently, more than 8800 such clones are known, with at least one clone on average per megabase (Mb) available on each chromosome. This resource affords the opportunity to generate an ordered array of DNA segments at very high genomic resolution and replace the metaphase spreads as the hybridization template. Such array-based CGH (A-CGH) circumvents the considerable limitations associated with the use of chromosome spreads. A high-resolution fluorescent scanner is utilized to capture the fluorescent intensity of each spot in the array and converts them into an intensity ratio. The fluorescence ratio of the two colors can be compared between different spots representing different genomic regions (Figure 7.1). This provides a genome-wide molecular profile of the sample with respect to regions of the genome that are deleted or amplified. The resolution level of this approach depends on a combination of the number, size, and map positions of the DNA elements within the array [6]. BAC arrays provide an opportunity to perform high-resolution genomic
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Array-Based Comparative Genomic Hybridization
Figure 7.1 (Color Figure 7.1 follows page 106.) The spectrum of genomic amplifications (green) and losses (red) in a tumor sample as detected by array-based CGH. The relatively high number of loci with color change indicates that the tumor has marked chromosomal instability. Each target clone is arrayed in triplicate and a high concordance can be seen among each of the adjacent replicate spots in the array.
scans in a rapid and highly reproducible fashion. Several array systems for CGH are available commercially (Spectral Genomics, Inc.; Vysis, Inc.). Alternatively, CGH can be performed on spotted cDNA arrays, allowing for direct comparison of the genomic changes with mRNA expression of the genes throughout the human genome. One of the commercially available platforms is the GenoSensor 300 from Vysis, Inc., Downer’s Grove, IL. The protocol discussed in this chapter is based on the authors’ experience with the latter system. 7.2 MATERIALS AND PROTOCOLS 7.2.1
DNA Isolation
It is very important to ensure that DNA of adequate amount, purity, and size is pre-
pared for microarray analysis. Depending on the tissue source, different kits and/or protocols might have to be used for DNA preparation. DNA that is heavily contaminated with other cellular components or is heavily degraded will not perform well in random priming or microarray hybridization. In particular, formaldehyde-fixed tissue may be problematic, and likely will require experimentation before extracted, labeled DNA appropriate for a DNA microarray analysis can be obtained. The Gentra Puregene DNA isolation kit is recommended for genomic DNA extraction. See the manufacturer’s catalog for proper kit selection and instructions for use (toll free: 888-476-5283; online ordering: www.gentra.com). For isolation of DNA from small amounts of cultured cells, we recommend the Puregene D-5500A (2 × 108 cells, 1 g tissue) or D-5000A (8 × 108 cells, 4 g tissue) kits.
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Molecular Morphology in Human Tissues: Techniques and Applications 7.2.2
DNA Quantitation
Once the DNA extraction has been completed and before moving onto the labeling protocol, the absorbance 260/280 must be measured or fluorometry reading taken to determine quantity and concentration of DNA. At least 100 ng of HMW good-quality DNA or 15,000 cells are required for the random labeling reaction. Once the DNA concentration is calculated, make a working solution of DNA at 25 ng/µl in 10 mM Tris, TE buffer, or 1 mM EDTA pH 7.4 to 8.0. Now you can continue on to the labeling protocol or store the DNA at −20°C. 7.2.3
DNA Labeling
Adequate DNA labeling is one of the most critical steps in microarray analysis. However, because of unavoidable varia-
7.2.3.1
tions in the quantity and quality of starting material, it is extremely difficult to specify labeling conditions that will work optimally in all situations. Therefore, the following protocol should be viewed only as a starting point, and it is strongly recommended that labeled DNA be checked on a gel prior to hybridization on a microarray. The following protocol has been found to reproducibly label 0.1 µg of reasonably intact extracted genomic DNA [7]. Different kits can be used to label DNA for use with the GenoSensor Array 300; however, the Vysis Random Priming Labeling kit has been optimized for the GenoSensor System and is recommended. The kit is available for online ordering at http://www.vysis.com/O.asp?ProductID= 349. All the instruments and supplies are listed in Section 7.2.3.1.
List of Reagents, Supplies, and Instrumentation for DNA Labeling
Random Priming Labeling Kit (Vysis, Downers Grove, IL) includes: 2.5× Random Priming Mix Klenow solution DNase reaction buffer GenoSensor Array 300 nucleotide mix Male and female reference DNA DNase dilution buffer
DNase I Amp grade Precipitation Reagent TE buffer Stop buffer 3 M sodium acetate 10 mM Tris
Further reagents include: Reagent
Company, Location, Order Number, and Web Site Address
1 mM cyanine 3-dCTP (PerkinElmer/NEL576)
Perkin-Elmer Life Sciences, Inc. Boston, MA Product No. NEL576001EA http://www.perkinelmer.com/nucleotide_analogs
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Array-Based Comparative Genomic Hybridization Reagent
Company, Location, Order Number, and Web Site Address
1 mM cyanine 5-dCTP (PerkinElmer/NEL577)
PerkinElmer Life Sciences, Inc. Boston, MA Product No. NEL577001EA http://www.perkinelmer.com/nucleotide_analogs
Absolute ethanol
AAPER Alcohol & Chemical Co. Shelbyville, KY
MicroSpin™ S-200 HR Columns (optional)
Amersham Biosciences Piscataway, NJ Cat. No. 27-5120-01 http://www.amersham.com
E-Gel® 2% Agarose
Invitrogen™ Life Technologies. Carlsbad, CA http://www.invitrogen.com
50 bp ladder
Life Technologies Cat. No. 10416-014
Supplies and instrumentation Reagent
Company, Location, Order Number, and Web Site Address
37°C incubator
Precision Scientific Inc. Chicago, IL
15°C, 37°C, and 100°C baths
Precision Scientific Inc. Chicago, IL
4°C microcentrifuge
Brinkmann Instruments, Inc. Product No. 0013 595 208-00
RNase/Dnase-free 1.7-ml microcentrifuge tubes
Costar™,Corning Inc. Corning, NY Cat. No. 3620
10 ml, 20 ml, 200 ml, and 1000 ml
Pipettors Rainin, Oakland, CA Cat. No. P10, P20, P200, P1000 adjustable pipetman http://www.rainin.com/products/product_list.asp?class=50
DNAse-free aerosol-resistant pipette tips
Rainin, Oakland, CA Cat. No. GP-10F, GP-20F, GP-200F, GP-1000F http://www.rainin.com/products/product_list.asp?class=150
Vortex mixer
Barnstead International Dubuque, IA Maxi Mix II
Ice bucket Powder-free gloves
Kimberly-Clark Co. Roswell, GA http://www.kchealthcare.com
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Molecular Morphology in Human Tissues: Techniques and Applications 7.2.3.2 Preparation of the Random Priming Reactions Because random priming is amplification reaction, the aerosol-resistant pipette tips and the ShaftGard aerosol-resistant tips will help avoid sample contamination with foreign DNA. In 1.7 ml Dnase-free tubes, mix the following reagents for test and reference labeling reactions: Reagent
Test
Reference
1.TE buffer 41.6 µl 41.6 µl 40 µl 2. 2.5 Random Priming Mix 40 µl – 3. Test DNA 4 µl 4. Sex-matched reference DNA – 4 µl 5. Denature at 100°C for 10 min. 6. After denaturing, immediately place the tubes on ice for 10 min. 7. Spin down the condensate inside the tubes 8. Place the tubes back on ice and add the following reagents.
7.2.3.3
DNase Digestion
Set up DNase Reactions and 1:20 DNase I Amp Grade dilution on ice. Dilute DNase Amp Grade 1:20 using DNase Dilution Buffer provided (make immediately before use and discard unused portion of the diluted DNase). Reagent
Test Reference
1. DNase Reaction Buffer 17 µl 17 µl 3 µl 2. 1:20 Diluted DNase Amp Grade 3 µl 3. Incubate at 15°C for 1 hr in the dark. 4. Immediately following DNase digestion, place the tubes on ice. 5. Quench reactions with 6 µl of stop buffer and then vortex.
7.2.3.4 1.
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Labeled Probe Purification
Prepare two Microspin S-200 HR columns as follows:
- Vortex the columns to resuspend the resin. - Loosen the cap a quarter turn and snap the bottom closure. - Place the column in a centrifuge tube for support (cut the caps of the tubes). - Pre-spin the columns at 3000 rpm for 1 min. - Place the column in a new tube, remove and discard the cap, and slowly apply the sample (126 µl of T and R tubes) to the top center of the resin, being careful not to disturb the bed. - Spin the column at 3000 rpm for 2 min. - Discard the column. 2. Add 12 µl of 3 M sodium acetate, (0.1 volume). 3. Add 1 µl precipitation reagent, then vortex briefly. 4. Add 350 µl of −20°C 100% ethanol, (2.5 volumes); vortex briefly. 5. Incubate at −20°C for 1 hr. 6. Centrifuge at 14,000 rpm for 30 min at 4 °C. 7. Remove the supernatant and then airdry pellets until no visible liquid is present in the tube. 8. Resuspend pellets in 4 µl of 10 mM Tris, pH 8. Gently vortex to facilitate resuspension. 9. Incubate at RT for 30 min prior to storage. 10. Quickly spin the tubes to collect the sample at the bottom of the tube. 11. Probe can be stored at −20°C if not used immediately for a gel analysis or hybridization reaction. 7.2.4
Hybridization
Different kits can be used for microarray hybridization, yet the Vysis GenoSensor
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Array-Based Comparative Genomic Hybridization Array 300 Hybridization Kit has been optimized for the GenoSensor 300.
7.2.4.2 Preparation of the Hybridization Mixture (One Chip)
GenoSensor array 300 Hybridization Kit (store at 15 to 25°C), Vysis, Inc. (Downers Grove, IL), Cat. No. 32-801040. For online ordering, http://www.vysis.com, or call toll-free: 800-553-7042.
1.
The kit contains five microarrays, hybridization coverslips, coverslips 18 mm, 20X SSC, and NP-40. Also, the microarray hybridization buffer and array DAPI are available from Vysis, Inc. (Downers Grove, IL). Cat. No. 32-803824. Further requirements are mainstays of the typical molecular pathology laboratory and include vortex mixer; 37°C incubator or heating block; 45°C slide warmer; 58°C and 80°C waterbaths; Coplin jars with lids (Wheaton, #900570); fine-tip forceps; 1.7-ml microcentrifuge tubes; 2-ml, 20-µl, and 200-µl pipettors; formamide (Sigma, St. Louis, MO; Product No. F-7508); compressed filtered air; and powder-free gloves (KimberlyClark Co., Roswell, GA; http://www.kchealthcare. com). 7.2.4.1 Preparing the Hybridization Solution Pre-warm the hybridization buffer to 37°C for 30 min, vortex and spin prior to use to ensure that the hybridization buffer is well dissolved. Due to the high concentration of Cot-1 DNA in hybridization buffer, the solution may appear cloudy or opalescent. Some cloudiness in the buffer after pre-warming is acceptable as long as there are no visible clumps of precipitate. Quickly spin the tube to collect the solution at the bottom of the tube. Hold the hybridization buffer at 37°C until immediately before use.
Combine the following in a 1.7-ml micrcentrifuge tube: a. Microarray hybridization buffer, 25 µl b. Test DNA, 2.5 µl c. Reference DNA, 2.5 µl 2. Vortex and quickly spin the sample. Note: Prior to addition, the probes should be centrifuged at 12,000 to 16,000 rpm for 1 min. When pipetting the probe, avoid any precipitate that may form at the bottom of the tube. 7.2.4.3
Hybridization Protocol
1.
Place the microcentrifuge tube containing the hybridization mixture into an 80°C water bath for 10 min to denature the DNA. 2. Remove from the water bath and centrifuge at 12,000 to 16,000 rpm for 5 sec. 3. Quickly transfer the microcentrifuge tube containing the hybridization mixture to a 37°C incubator or covered heating block; incubate in the dark for 1 hr. 4. Remove the necessary number of microarrays from their protective packaging using the tear marks. Place the microarrays in a 37°C dry air incubator for 30 min prior to use. 5. Place a filter paper folded in quarters on the bottom of a sealable box. Add enough of the 50% formamide/2X SSC wash solution to saturate the filter paper. Note: Saturation with any other solution will cause hybridization failure. 6. Place the box in 37°C dry air incubator for 30 min to 1 hr prior to use. Note: Complete Steps 7 through 9 on one array before beginning the next array. Do 129
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Molecular Morphology in Human Tissues: Techniques and Applications not allow the tubes to sit at room temperature. If multiple hybridizations are to be set up at one time, use a 37°C heating block to hold the tubes. 7. Carefully remove the cover protecting the microarray hybridization area and discard. 8. Remove the tube containing the hybridization solution from the 37°C heating block. Mix gently and quickly spin. Draw the full amount of hybridization mixture into a pipette tip and slowly add the hybridization mixture onto the corner of the array. Be very careful not to touch the pipette tip on the DNA array area or introduce air bubbles (leave the microarray on the slide warmer). 9. Place the microarray in the prewarmed box in 37°C incubator. 10. Repeat Steps 5 through 9 for the remaining microarrays. 11. Hybridize arrays for 60 to 72 hr. 7.2.4.4
Washing the Microarrays
Preparing the washing solutions: 20× SSC: Mix thoroughly 66 g (entire bottle) of 20× SSC in 250 ml purified H2O. Store at ambient temperature no longer than 6 months. 1× SSC/0.1% NP40: Pre-warm NP-40 to 58 ± 1°C. Mix 50 ml 1× SSC (prepared as described above) with pre-warmed 50 ml NP-40 (using wide-orifice tips will facilitate pipetting of the viscous liquid). Mix gently, making sure that the detergent is completely dissolved. Pour enough wash solution into a Coplin jar to cover the arrays but not so much as to immerse the labels on themicroarray holder; label the jar “1.” This wash should be discarded after use. 0.1× SSC/0.1% NP-40: Pre-warm NP40 to 58 ± 1°C. Add 5 ml of 1× SSC to 45 ml purified H2O. Add 50 ml pre-warmed NP-40 (using wide-orifice tips will facili130
tate pipetting of the viscous liquid) to 50 ml of 0.1× SSC. Mix gently, making sure that the detergent is completely dissolved. Label a glass Coplin jar with lid, “2.” Pour enough wash solution into the jar to cover the arrays but not so much as to immerse the labels on the microarray holder. Discard washes after use. 1× SSC: Pour enough wash solution (prepared as described above) into a Coplin jar to cover arrays but not so much as to immerse the labels on the microarray holder, label the jar “3.” This wash should be discarded after use. It must be mentioned that the washing step is very sensitive and from our as well as others’ experience, we have provided here some technical hints to help ensure proper washing. It is not recommended to wash more than five microarrays at the same time. Optimally, washes should be done in reduced light if possible. Adequate washing is vital to assay performance. Therefore, microarrays should be agitated with a backand-forth motion (approx. ten times), prior to transferring from one bath to the next. The microarray should not be allowed to dry at any step upon completion of the hybridization or after the final rinse. To not to allow the microarrays to dry between washes, always move chips quickly from one wash to another without examining them and avoid exposing them to high airflow when processing the arrays. Never apply force when washing the chips. Avoid twisting the arrays in the Coplin jars and be careful when moving the chips up and down in the grooves of the jars, so as to prevent cracking or dislodging of the chip from its holder. Now proceed with the washing steps:
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Array-Based Comparative Genomic Hybridization 1.
2. 3.
4. 5.
6.
7.
8. 9.
Place 1× SSC/0.1% NP40 washes (#1) in a 58°C water bath, allowing them to equilibrate for approximately 30 min. Prior to washing arrays, use a thermometer to check washes are at 58 ± 1°C. Place a Coplin jar containing 1× SSC (#3) and another containing dH2O (#4) at room temperature. Remove the microarrays from the box in the incubator, and using a fine forceps, remove the hybridization coverslip by grabbing the overhanging edge and gently lifting up. Place up to four slides QUICKLY in jar #1 at 58°C. The temperature should remain >55°C. Incubate for 4 min, mix by gently moving each slide up and down five times every 1 min. Do not take the slide out of the solution. Agitate each slide briefly, and quickly transfer each slide to jar #2 at 58°C; incubate for 4 min and do the same as in Step 5. Agitate each slide briefly, and quickly transfer each slide to jar #3 at room temperature, incubate for 1 min, agitate slide, transfer to water rinse. Rinse in d H2O for 1 to 2 sec. Briskly shake the chip twice to get rid of the excess water, thus ensuring that the array area itself remains wet.
7.2.4.5 Applying the Mounting Solution and Coverslip Allow the array DAPI to warm to room temperature prior to pipetting. 1.
Remove an 18 × 18-mm coverslip (provided in the kit) from the box, being careful not to touch either surface at any time. Remove any dust or lint from both sides.
2.
Pipette 20 µl array DAPI solution onto the coverslip, turn it over, and carefully place it over the wet microarray by inserting it into the recessed area. (Do not substitute with other DAPI solutions.)
To avoid air bubbles, it is best to start at an angle at one side of the microarray and allow it to fall slowly onto the microarray. Do not tap or squeeze the coverslip as this may damage the target spots on the microarray surface. Store the hybridized microarrays in the dark (preferably in a box with an airtight lid) for 45 min prior to imaging. 7.2.4.6
Visualizing the Hybridization
Under most circumstances, auto-exposure should be tried first. Certain conditions may warrant manual adjustment of the exposure settings to achieve optimal results. For example, if targets with very high amplification levels are detected, a subsequent longer exposure may be required (which will overexpose the highlevel amplifications) to achieve good intensity levels on the unamplified targets. Similarly, if the image contains a significant amount of brightly fluorescent debris, manual adjustment of the exposure (and subsequent editing of the image) may be necessary to achieve adequate signal levels. The supplied Vysis high-resolution scanner with a large-field multicolor fluorescence imaging system captures an image of the hybridized chip in three color planes: Cy 3, Cy5, and DAPI blue. The built-in software automatically identifies each spot, analyzes the Cy3/C5 ratios on all targets, and calculates the ratio most representative of the modal DNA copy number of the sample DNA. For each target, the normalized ratio, relative to the modal DNA copy number, is calculated.
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Molecular Morphology in Human Tissues: Techniques and Applications This normalized ratio of a target indicates the degree of gain or loss of copy number compared with the sample’s modal copy number. Ratios above 1.2 indicate genomic gain and ratios below 0.8 indicate genomic loss, and only changes with significant p-values should be considered reliable (60%), cells are washed gently with 1× PBS, heat fixed, and then fixed with 2% paraformaldehyde, overnight.
http://www.chemie.uni-marburg.de/ ~becker/welcome.html
12.3.1.3
http://www.mcb.uct.ac.za/pcroptim.htm http://www.premierbiosoft.com/primerdesign/index.html 12.3 PRACTICAL PROTOCOLS This section deals with the practical aspects of in situ PCR. 12.3.1 Preparation of Tissues 12.3.1.1 Cell Suspensions To use peripheral blood leukocytes, first isolate cells on a Ficoll-Hypaque density gradient. Tissue-culture cells or other single-cell suspensions can also be used. Prepare all cell suspensions with the following procedure: 1. 2. 3.
4.
Wash cells with 1× PBS twice. Resuspend cells in PBS at 2 × 106 cells/ml. Add at least 10 µl cell suspension to each well of the slide using a P20 micropipet spread across surface of slides. Air-dry slide in a laminar-flow hood.
12.3.1.2
Adherent Cultured Cells
There are several types of slides designed to support in situ PCR after they are attached on the glass slides. The cells are grown on these types of slides. If certain primary cell cultures require attachment 190
Paraffin-Fixed Tissue
Routinely fixed paraffin tissue sections can be utilized for amplification purpose quite successfully. This permits the evaluation of individual cells in the tissue for the presence of a specific RNA or DNA sequence. For this purpose, tissue sections are placed on routine histological slides. Tissue sections should be sliced to a 5- to 6-µm thickness. However, if one is using tissues that contain particularly large cells (such as ovarian follicles), then thicker sections may be appropriate. Before in situ PCR, the slides must be deparaffinized. For this purpose, follow the following protocol: 1.
2.
3.
Incubate the slides in an oven at 60 to 80°C (depending on the type of paraffin used to embed the tissue) for 1 hr to melt the paraffin. Dip the slides in EM grade xylene solution for 5 min, then in EM grade 100% ethanol for 5 min (EM grade reagents are benzene-free). Repeat these washes two or three times to completely rid the tissue of paraffin. Dry the slides in an oven at 80°C for 1 hr.
12.3.1.4 Creating Micro-Well for In Situ PCR Before the actual amplification process can be initiated, we have to create a vaportight sealing chamber (M.J. Research, www.mjr.com) that can withstand the high temperature required for PCR. Once an artificial well is created, one can place the
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Recent Developments in In Situ PCR PCR solution so that the putative genes of interest can be amplified in situ. For this purpose we can use “Frame-Seal Incubation Chambers.” These frames have double-sided adhesive surfaces: one on the bottom that sticks to the surface of slides and one on the top that can be sealed by a plastic cover, after it has been placed in the correct amount of PCR solution on the tissue surface. To create an artificial well, the adhesive frame is first attached to the slide, enclosing the specimen area. Next, add the reaction cocktail and seal with the flexible plastic coverslip. This vapor-tight chamber can withstand temperatures up to 99°C. After completing the amplification process, the entire artificially created well can be removed by simply pilling-off the adhesive frame. In addition, if one requires growing cells or small fragments of tissues inside the artificially created well, then the entire adhesive frame and the slide can be sterilized either by UV treatment or autoclaving the whole slide with frame, before seeding the cell cultures (see Figure 12.1 for details). 12.3.2 In Situ PCR 12.3.2.1 Basic Preparation: All Protocols For all sample types, the following steps comprise the basic preparatory work that must be performed before any amplification-hybridization procedure. The overview is depicted in Figure 12.2. 12.3.2.2
Figure 12.1 Frame-Seal Incubation Chamber for in situ PCR. On glass slides containing tissues or cells, a chamber is created utilizing the Frame-Seal Chamber. First, the double-sided adhesive frame is attached to the slide, enclosing the specimen area (steps 1 and 2). The reaction mix is then added and the reaction is sealed in place with the heat-resistant flexible plastic coverslip (steps 3 and 4). The gas-tight seal withstands temperatures up to 97°C. After thermocycling, the entire chamber can be removed from the slide with no residue remaining. The frame-seal can be UV-treated and is autoclavable (in case one is working with madcow agents etc.).
This step is absolutely critical. One may need to experiment with different periods to optimize the heat treatment for specific tissues. Our laboratory routinely uses 90 sec for DNA target sequences, and ~30 sec for the RNA sequences (for RT in situ PCR). 12.3.2.3 1.
Heat Treatment
Place the slides with the adhered tissues or cells on a heat-block at 105°C for 0.5 to 2 min to stabilize the cells or tissue on the glass surface of the slide.
2.
Fixation and Washes
Place the slides in a solution of 2% paraformaldehyde (PFA) in PBS (pH 7.4) for 2 hr at room temperature. Use of the recommended Coplin jars or staining dishes facilitates these steps. Wash the slides once with 3× PBS for 10 min, agitating periodically with an up-and-down motion.
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Molecular Morphology in Human Tissues: Techniques and Applications DNA Target Thin tissue sections, cell suspensions, cells cultured on slide, or chromosome spreads
RNA Target Sequence Same as for DNA reactions but reagents must be RNAse free (DEPC - treated)
Air dry, then heat @ 105°C for 90 – 120 sec
Air dry
2% paraformaldehyde overnight, wash once in 3x PBS then twice in 1x PBS
Same as DNA protocol
Proteinase K treatment (6µg/ml for 10 – 60 min, must be optimized)
Same as DNA protocol
Hydrogen peroxide treatment (optional)
Hydrogen peroxide treatment (1 hr)
See Figure 1 for Frame-Seal assembly
DNAse treatment to destroy genous DNA (may not be necessary if target sequence is spliced; see Figure 12.3)
Reverse transcription of RNA with appropriate primers In situ amplification in a thermal cycler (30 cycles, except for chromosome bands, which need 12–15 cycles) Seal with self-seal or slide frame Peel-off the Frame-Seal, wash in 2xSSC vPerform in situ hybridization with a tagged probe that anneals to an internal region of amplicons
Thermal Cyder (Twin-Tower)
Use probe-detection system, look for color in cytoplasm, nuclei, or bands, or count grains from radioactivity
Good practice allows signal to be contained within the cells
Figure 12.2 Overview of in situ DNA and RT PCRs.
3.
4.
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Wash the slides with 1× PBS for 10 minutes, agitating periodically with an up-and-down motion. Repeat once with fresh 1× PBS. At this point, slides with adhered tissues can be stored at −80°C until use. Before storage, dehydrate with 100% ethanol.
If biotinylated probes or peroxidasebased color developments are used, the samples should further be treated with a 3% solution of hydrogen peroxide in PBS to inactivate any endogenous peroxidase activity. Incubate the slides or store at room temperature for 10 to 20 min. Then wash the slides once with PBS.
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Recent Developments in In Situ PCR If other probes are to be used, proceed directly to the following proteinase K digestion, which is perhaps the most critical step in the protocol. 12.3.2.4 Proteinase K Treatment (The Most Rate Limiting Step) 1.
2. 3.
Treat samples with 6 µg/ml proteinase K in PBS for 5 to 60 min at room temperature for 5 to 10 min. To make a proper solution, dilute 1.0 ml proteinase K at (1 mg/ml) in 150 ml of 1× PBS. After proper digestion, heat slides on a block at 95°C for 2 min to inactive the proteinase K. Rinse slides in 1× PBS for 10 sec.
4. 5.
Rinse slides in distilled water for 10 sec. Air dry.
12.3.2.5 RT Variation: In Situ RNA Amplification One has two choices to detect an RNA signal. The first and more elegant method is to simply use primer pairs that flank spliced sequences of mRNA, as these particular sequences will be found only in RNA and be split into sections in the DNA (see Figure 12.3). Thus, using these RNA-specific primers, one can skip the following DNase step and proceed directly to reverse transcription.
Figure 12.3 Overview of the multiple splicing system and design for exon-specific primers. The majority of the eukaryotic genes are split into segments, as there are numerous introns (intervening segments) in the DNA (genome) that are not expressed but are excised during the post-transcription processing of RNA in the nuclear “spliceosome.” This characteristic can be exploited in designing of primers that can specifically amplify mRNA and not genomic DNA. One can simply design primers in such a manner that flank spliced regions where two exons (expressed genes) are fused, such that the complementary annealing sites exist only in mRNA and not in DNA. This allows the elimination of DNase treatment during slide preparation, as well as the simultaneous amplification of both RNA and DNA signals from the same gene.
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Molecular Morphology in Human Tissues: Techniques and Applications The second, more brutal, yet often necessary approach is to treat the cells or tissue with a DNase solution subsequent to proteinase K digestion. This step destroys the entire endogenous DNA in the cells so that only RNA survives to provide the signals for amplification. Note: All reagents for RT in situ amplification should be prepared with RNase-free water (i.e., DEPC-treated water). In addition, the silanized glass slides and all glassware should be RNase-free, which we ensure by baking the glassware overnight in an oven before use in the RT amplification procedure. 12.3.2.6 Reverse Transcriptase Reaction Next, one wants to make cDNA of the targeted RNA sequence so that the signal can be amplified. One should follow the manufacturer’s suggestion to prepare the cocktail. After preparing the RT-PCR solution, one should follow the following protocol: 1.
Add 10 to 15 µl of either cocktail to each well. Carefully peel the top of the frame seal and place the plastic covers on top of the frame seal section of the slide. Gently push with a curved forceps to make sure the cover is safely sealed. If using the rTth enzyme, program the thermocycler according to the manufacturer’s suggestion and proceed.
12.3.2.7 RT-Primers and Target Sequences In our laboratory, we simply use antisense downstream primers for our gene-ofinterest, as we already know the sequence of most genes we study. However, one can alternatively use oligo (dT) primers to first convert all mRNA populations into cDNA, 194
and then perform the in situ amplification for a specific cDNA. This technique may be useful when performing amplification of several different gene transcripts at the same time in a single cell. For example, if one is attempting to detect various cytokine expressions, one can use an oligo (dT) primer to reverse transcribe all the mRNA copies in a cell or tissue section. Then one can amplify more than one type of cytokine and detect the various types with different-colored probes that develop into different colors. In all RT reactions, it is advantageous to reverse-transcribe only relatively small fragments of mRNA (20 different fluorochromes are available), and DNA can be labeled with a biotin-peroxidase probe or a fluorochrome with a different-colored emission. Each will show a different signal within an individual cell [14, 22]. 12.3.2.11
Hybridization
The in situ hybridization (ISH) technique has been successfully applied in both research and clinical settings. However, a single, easy-to-use universal procedure has not been developed. Therefore, the specific needs of the diagnostic or research goals must be considered when choosing a suitable protocol. We will leave this up to each investigator to determine his or her own optimal protocol. 12.3.2.12
Controls
The validity of in situ amplificationhybridization should be examined in every run. Attention here is especially necessary in laboratories first using the technique because occasional technical pitfalls lie along the path to mastery. In an experienced laboratory, it is still necessary to continuously validate the procedure and to confirm the efficiency of amplification. To do this, we routinely run two or three sets of experiments in multi-welled slides simultaneously, for we must not only validate amplification, but also confirm the subsequent hybridization/detection steps. In our lab, we frequently work with infectious agents. A common validation 196
procedure we conduct is to mix infected cells with uninfected cells in known ratios (e.g., 1:10, 1:100, etc.), and then confirm that the results are appropriately proportionate. To examine the efficiency of amplification, one can use a cell line, which carries a single copy or two copies of the gene of interest [1–14], and then look to see that proper amplification and hybridization have occurred. In all amplification procedures, we use one slide as a control for nonspecific binding of the probe. Here we hybridize the amplified cells with an unrelated probe. We also use HLA-DQa and beta-actin probes and primers with human peripheral blood mononuclear cells (PBMC) and other tissue sections as positive controls to check the various parameters of our system. If one is using tissue sections, a cell suspension lacking the gene-of-interest can be used as a control. These cells can be added on top of the tissue section and then retrieved after the amplification procedure. The cell suspension can then be analyzed with the specific probe to see if the signal from the tissue leaked out and entered the cells floating above. We suggest that researchers carefully design and employ appropriate positive and negative controls for their specific experiments. In the case of RT in situ amplification, one can use beta-actin, gamma-globulin, HLA-DQa, and other endogenous-abundant RNAs as the positive markers. Of course, one should always have an RT-negative control for RT in situ amplification, as well as DNase and nonDNase controls. Controls without polymerase plus primers and without primers should always be included. (See Figure 12.4.)
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Figure 12.4 (Color Figure 12.4 follows page 106.) DNA in situ PCR analysis of a “293” cell line infected by co-culture with HHV-8-infected motile sperms in vitro. The presence of episomal HHV-8 virions in these cells could be visualized in about 10 to 15% of the cells (yellow-green). Sperm from an HHV-8-infected man showing positive staining for the virions at the tails, midpieces, and heads of the sperm.
References 1. 2.
3.
4.
5.
6. 7.
Bagasra, O., Polymerase Chain Reaction In Situ. Amplifications, Editorial note, 20, 1990. Pestaner, J.P. et al., Potential of in situ polymerase chain reaction in diagnostic cytology, Acta Cytologia, 38, 676, 1994. Patterson, B. et al., Detection of HIV-I DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry, Science, 260, 976, 1993. Maggioncalda, J. et al., A herpes simplex virus mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics, J. NeuroVirology, 2, 268, 1996. Sullivan, D.E. et al., Self-seal reagent: evporation control for molecular histology procedure without chambers, clips or fingernail polish, BioTechniques, 23, 320, 1997. Winslow, B.J. et al., HIV-1 latency due to the site of proviral integration, Virology, 196, 849, 1993. Bobroski, L. et al., Localization of human Herpes virus type 8 (HHV-8) in the Kaposi’s sarcoma tissues and the semen specimens of HIV-1 infected and uninfected individuals by utilizing in situ polymerase chain reaction, J. Reprod. Immunol., 41, 149, 1999.
8.
9.
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Embretson, J. et al., Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution, Proc. Natl. Acad. Sci. U.S.A., 90, 357, 1993. Embretson, J. et al., Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS, Nature, 62, 359, 1993. Ewida, A.S., Raphael, S., and Bagasra, O., The presence of IL-2 and IL-10 cytokines in the skin lesions of Blau Syndrome, Appl. Immunochem. Mol. Morphol., 10, 171, 2002. Harrington, W., Jr. et al., Human Herpes virus 8 (HHV-8) DNA sequences in cell free plasm and mononuclear cells in AIDS and non-AIDS patients, J. Infect. Dis., 174, 1101, 1996. Nuovo, G.J., PCR In Situ Hybridization Protocols and Applications, 2nd ed., Raven Press, New York, 1994. Bagasra, O. et al., Detection of HIV-1 provirus in mononuclear cells by in situ PCR, New Engl. J. Med., 326, 1385, 1992. Bagasra, O., Seshamma, T., and Pomerantz, R.J., Polymerase chain reaction in situ: intracellular amplification and detection of HIV-1 proviral DNA and other specific genes, J. Immunol. Meth., 158, 131, 1993.
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Molecular Morphology in Human Tissues: Techniques and Applications 15. Bagasra, O. et al., High percentages of CD4-positive lymphocytes harbor the HIV-1 provirus in the blood of certain infected individuals, AIDS, 7, 1419, 1993. 16. Bagasra, O. et al., Human immunodeficiency virus type 1 infection of sperm in vivo, AIDS, 8, 1669, 1994. 17. Bagasra, O. et al., Activation of the inducible form of nitric oxide synthetase in the brains of patients with multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A., 92, 12041, 1995. 18. Bagasra, O. and Pomerantz, R.J., Human herpesvirus 8 DNA sequences in CD8+ T-cells, Letter, J. Infect. Dis., 176, 541, 1997. 19. Bagasra, O. et al., In situ polymerase chain reaction and hybridization to detect low abundance nucleic acid targets, in Current Protocols in Molecular Biology, Ausubel et al., Eds., Wiley Interscience, New York, 1995, chap. 14. 20. Bagasra, O. et al., Activation of the inducible form of nitric oxide synthetase in the brains of patients with multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A., 92, 12041, 1995. 21. Bagasra, O. et al., Cellular reservoirs of HIV-1 in the central nervous system of infected-individuals: identification by the combination of in situ PCR and immunohistochemistry, AIDS, 10, 573, 1996. 22. Bagasra, O. and Amjad, M., Protection against retroviruses are owing to a different form of immunity: an RNA-based molecular immunity hypothesis, Appl. Immunohistochem. Mol. Morphol., 8, 133, 2000. 23. Bobroski, L. et al., Mechanism of vertical transmission of HIV-1: role of intervillous space, Appl. Immunohistochem. Mol. Morphol., 7, 271, 1999.
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24. Pellett, P.E. et al., Multi-center comparison of polymerase chain reaction detection of human Herpesvirus 8 DNA in semen, J. Clin. Microbiol., 37, 1293, 1999. 25. Qureshi, M.N. et al., Localization of HIV-1 proviral DNA in oral mucosal epithelial cells, J. Infect. Dis., 171, 190, 1994. 26. Strayer, D.S. et al., Titering replication-defective virus for use in gene transfer, BioTechniques, 22, 447, 1997. 27. Pereira, R.F. et al., Cultured stromal cells from marrow serve as stem cells for bone, lung and cartilage in irradiated mice, Proc. Natl. Acad. Sci. U.S.A., 92, 4857, 1995. 28. Hooper, D.C. et al., Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A., 94, 2528, 1997. 29. Lattime, E.C. et al., Expression of cytokine mRNA in human melanoma tissue, Cancer Immunol. Immunother., 41, 151, 1995. 30. Hsu, T.-C. et al., Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI, J. Biol. Chem., 273, 13787, 1998. 31. Rishi, I.R. et al., Down-regulation of hZIP1 and hZIP2 zinc transporters in the prostate cancer tissues from African decent as compared to Caucasian men, Appl. Immunohistochem. Mol. Morphol., 10, 171, 2003. 32. Bagasra, O. and Hansen, J., In Situ PCR Techniques, John Wiley & Sons, New York, 1997.
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High-Resolution Visualization of Apoptotic Markers in Human Biopsies Andreas P. Aschoff and Gustav F. Jirikowski
13.1 INTRODUCTION For the demonstration of apoptotic cells in tissue sections, histological detection of DNA fragmentation by in situ end labeling (ISEL) has been widely used. With this method, apoptotic cells can be easily detected, even in cases were only single cells are dying. In contrast, it is not easy to detect the morphological changes in cells undergoing apoptosis, such as chromatin condensation and cell shrinkage, especially in cases were only a few cells are involved. Although DNA fragmentation can be demonstrated in apoptotic cells and apoptotic bodies in most cases, there is no clear correlation of ISEL staining with apoptosis, because in many morphologically intact cells, nuclei with fragmented DNA can be found [1]. Such non-apoptotic cells can be labeled satisfactorily with methods for the detection of fragmented DNA in situ [2–5]. Thus, staining with ISEL for the detection of apoptosis is only useful when characteristic morphological changes or biochemical markers for apoptosis are likewise considered. In a search for unequivocal markers for apoptotic cells, we tried to 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
localize proteins known to be involved in programmed cell death within cells showing different degrees of DNA fragmentation. Semi-thin tissue sections of the small intestine were chosen, in an effort to demonstrate a gradient of DNA damage along the crypt-villus axis [1, 6]. One protein, the enzyme tissue transglutaminase (TTG) or transglutaminase type II, is expressed and activated in tissues where many cells are found that supposedly undergo apoptosis as judged by morphology and/or DNA fragmentation [7–13]. Although DNA fragmentation and expression of TTG are often increased in tissues with an elevated apoptotic index [8], the co-localization of DNA fragmentation and TTG protein in apoptotic cells has not yet been demonstrated. Because expression of TTG protein is also involved in differentiation, growth, and adhesion of cells [14], the occurrence of TTG protein alone is probably not sufficient for demonstrating apoptosis in all circumstances and cell types [15, 16]. We have shown that the protein TTG is unequivocally expressed in apoptotic enterocytes [17] and other apoptotic cells
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Molecular Morphology in Human Tissues: Techniques and Applications (e.g., in human heart [18], both by DNA fragmentation and morphology). TTG is not expressed in intact cells, although in many cases DNA fragmentation can be demonstrated. Thus, the immunohistochemical demonstration of TTG may serve as a simple marker for apoptotic epithelial cells in tissue sections when co-localized with DNA fragmentation markers.
embedding methods and cutting semithin sections from biopsies taken from different sites of the organ studied, both limitations can be overcome to a large extent.
The use of biopsy material for the detection of apoptosis and the study of apoptotic regulation is preferable to postmortem tissue sections for many reasons. Due to rapid fixation, biopsy materials normally have excellent structural preservation, and post-mortal enzymatic and biochemical changes cannot occur. Thus, in many cases of human tissue obtained after a rather long post-mortem delay and not by biopsies, false positive DNA fragmentation has been found, due to enzymatic digestion of DNA [1]. Another advantage to using biopsy material is the fact that tissue samples can be taken in different stages of illness and then compared with healthy tissue from the same patient. Thus, the progress of illness can be monitored and, likewise, the effect of various treatments can be monitored without serious operations. One problem with biopsy material is the fact that focal changes or variations of tissue integrity within the organ are difficult to detect. This can only be overcome by taking biopsies from various regions of the organ. Another limitation in the use of biopsy material is the size of the tissue sample. Thus, in routine histological processing and sectioning, only a few sections can be taken from one biopsy sample. This may present problems when several different markers must be studied, because simultaneous detection of different antigens in the same section can be a relatively difficult task. Using special
Biopsies of human duodenum or human kidney were removed and immediately fixed in 4% buffered paraformaldehyde for 24 hr. After dehydration through an ascending alcohol series, they were embedded in Epon resin.
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13.2
MATERIAL AND REAGENTS
13.2.1
Fixation
13.2.1.1 Preparation of Buffered Paraformaldehyde To prepare 1000 ml of the buffered paraformaldehyde solution, we recommend the following procedure: 1.
2.
3.
4.
Prepare a 0.2 M sodium dihydrogen orthophosphate solution (NaH2PO4, MW 156) by adding 31.20 g to 1000 ml distilled water. Prepare a 0.2 M disodium hydrogen orthophosphate solution (Na2HPO4, MW 142) by adding 28.4 g to 1000 ml distilled water. 140 ml NaH2PO4 solution are added to 360 ml Na2HPO4 solution, resulting in a 0.2 M phosphate buffer with pH 7.4. This buffer (stored at 4°C) should be used to prepare the buffered paraformaldehyde. Preferably in a fume cupboard, add 40 g paraformaldehyde (a white powder) to 400 ml distilled water. Heat, while stirring, to 60°C. When the paraformaldehyde is dissolved, take the beaker away from the heater and let it cool. When the paraformalde-
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High-Resolution Visualization of Apoptotic Markers in Human Biopsies hyde solution has reached room temperature, add 500 ml of 0.2 M phosphate buffer. Add as much distilled water as necessary to bring the total volume to 1000 ml. Technical Hints: The pH can, if necessary, be adjusted by adding drops of either HCl or NaOH. Never use commercially available formaldehyde solutions. These are normally not stabilized and contain formic acid, which could impair the fixation and therefore make it unreliable for the technique described here. If the paraformaldehyde solution does not become clear during heating, add drops of NaOH. 13.2.1.2
Reagents
NaH2PO4·2H20 (monosodium-phosphate, puriss.) Fluka 04270 Na2HPO4 (disodiumhydrogenphosphate, puriss.) Fluka 71640 Paraformaldehyde, puriss. Fluka 16005 NaCl, puriss. Fluka 71382 NaOH Fluka 34282 13.2.2 Dehydration Dehydration should be performed after 24 hr of fixation. Longer fixation times are possible. For dehydration, place the tissue blocks in a vessel with at least 50 ml ethanol, and begin with a concentration of 50% ethanol for at least 30 min. Longer dehydration periods are not harmful. Repeat the dehydration steps for at least 30 min with concentrations of 70, 80, and 90%. ethanol. To complete dehydration, place the specimen in 95% ethanol and change the ethanol three times. In 95%
ethanol, the specimen should not rest longer than 3 hr. 13.2.3 Embedding in Epon 13.2.3.1 Embedding Procedure To embed the dehydrated material, place the specimens in propylenoxide for 15 min and change the solution twice (2× propylenoxide, 15 min each). After propylenoxide clearing, place the specimens in Epon C mixture, which is prepared freshly as follows: Epon A: 19 g Epon 812 + 31 g DDSA (together 50 g) Epon B: 24 g Epon 812 + 26 g MNA (together 50 g) Epon C: Mix Epon A and Epon B thoroughly, and add 800 ml propylenoxide. Again, mix thoroughly. For embedding, place the specimens into the Epon C mixture (= “first embedding mixture”) using an open beaker. Place the beaker under the hood and let the propylenoxide evaporate for 24 hr. After 24 hr in Epon C mixture, place the samples in the “second embedding mixture” (Epon D), consisting of Epon A (50 g) + Epon B (50 g) + 2 g DMP-30 (“accelerator”). Leave the specimens in Epon D for at least 1 hr. Now prepare a fresh “third embedding mixture” like Epon D. Fill this mixture in the embedding molds. Then place the specimen in the embedding mold, orientate, and incubate for 3 days at 65°C in an oven. After this hardening process of the Epon resin, the embedding procedure is finished, and small blocks can be cut for sectioning. 201
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Molecular Morphology in Human Tissues: Techniques and Applications 13.2.3.2 Reagents for Epon Embedding Propylenoxide (1,2-epoxy-propane), puriss. Fluka 82320 Epon E 812 Fluka 45345 DMP-30 (accelerator) Fluka 45348 MNA (Methyl nadic anhydrid, hardener) Fluka 45347 DDSA (hardener) Fluka 45346
13.3 SECTIONING AND PREPARATION OF SLIDES Tissue blocks were sectioned on a Leica-Reichert Ultracut microtome (Leica, Wetzlar, Germany) with standard glass knives into serial semi-thin sections, 0.5 to 1.0 µm thick. Sections were collected in the knife’s water bath and transferred with a small glass rod onto water drops at the surface of the microscopic slides (“superfrost” slides). They were dried on a hotplate at 80°C and then stored until further use. Technical Hint: Tissue embedded in Epon proved stable for long periods of time. Even nucleic acids seem to be well preserved after several years, as could be shown with in situ hybridization for various mRNA sequences [91]. 13.4 STAINING PROCEDURE 13.4.1 13.4.1.1
Removing the Resin Procedure
Prior to histochemical staining, resin must be removed from semi-thin sections with a sodium methoxide solution (Solution A). Slides are incubated for 2 min in 202
the solution, followed by incubation for 3 min in a 1:1 methanol–benzene mixture, followed by acetone (2×, 3 min each), and stored in 66 mM PBS (Solution B). The sections are now ready for staining. Technical Hint: The sodium methoxide solution as well as the mixture of methanol and benzene can be reused many times, while acetone and PBS must be discarded after single use. Preparation of Sodium-Methoxide (Solution A): Sodium metal (10 g) is dissolved in 100 ml of 100% methanol overnight in a hood. Benzene (20 ml) is slowly added while stirring the mixture. The solution is stable for several years at room temperature if stored in a closed, brown glass bottle, and it can used repeatedly. Sodium methoxide solution must be replaced when it changes from a clear color to brown and contains a lot of precipitates. Technical Hint: Treating the Epon sections with sodium methoxide should take as long as necessary but as short as possible. Therefore, test sections should be immersed for either 1, 2, 3, 4, or 5 min in the sodium methoxide solution and then stained with Sudan Black (1% in 70% ethanol). Because Epon is stained by this dye, this technique allows the monitoring of resin removal. When the optimal time for resin removal has been determined, all other sections can be treated simultaneously with the same time schedule. Preparation of Phosphate-Buffered Saline (PBS, Solution B): To prepare 66 PBS, dissolve 9.32 g mM Na2HPO4·2H2O, then 1.8 g KH2PO4, and then 9 g NaCl in 1000 ml distilled water. The pH should be 7.3.
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Reagents for Resin Removal
Na2HPO4·2H2O KH2PO4 (potassium phosphate, monobasic) NaCl Benzene Methanol Acetone Metallic sodium Sudan Black B 13.4.1.3
Fluka 71640 Sigma P5379 Fluka 71382 Fluka 12552 Fluka 65543 Fluka 00570 Fluka 13401 Sigma S2380
Suppliers
Sigma: www.sigma-aldrich.com Fluka Chemie (now belongs to SigmaAldrich): www.sigma-aldrich.com 13.4.2 13.4.2.1
In Situ Tailing with BrdU Procedure
After rinsing the sections in 66 mm PBS, they are incubated with TdT reaction mixture at 37°C for 1 hr. TdT reaction mixture: Mix 4 µl (e.g., 100 Units) of terminal transferase (TdT), with 8 µl TdT reaction buffer, 3 µl of 25 mM cobalt chloride solution (CoCl2 solution), and 1 µl of 25 mM BrdU solution with 400 µl of 67 mM PBS. 13.4.2.2 Reaction
Reagents for the BrdU
Terminaltransferase Roche 3333566 TdT reaction buffer Roche 3333566 Roche 3333566 CoCl2 Bromodeoxyuridine (5′-bromo-2′-deoxyuridine) Sigma B9285
13.4.2.3
Suppliers
Sigma: www.sigma-aldrich.com Roche Diagnostics: Sandhoferstr. 116, D-68305 Mannheim; www.rocheapplied-science.com Technical Hints: TdT is very expensive and must be stored at −20°C. We therefore buy TdT in bulk packages (10,000 units) and prepare small samples of about 500 units (20 µl). To increase the sensitivity of the method, it is useless to utilize larger amounts of TdT or to prolong the incubation time. The sensitivity of detection and/or staining intensity can be controlled by the amount of BrdU used. When more BrdU is added, sensitivity can be greatly increased. 13.4.3 BrdU
Immunohistochemistry for
13.4.3.1 Procedure Primary Antibody: After incubation with the TdT reaction mixture, sections are washed in PBS and then incubated with the primary antibody (monoclonal mouse anti-BrdU), diluted 1:100 in PBS with 2% normal goat serum, at 4°C for 12 hr in a wet chamber. Thereafter, sections are washed two times in PBS for at least 10 min each. Secondary Antibody: Incubate with goat anti-mouse IgG (diluted 1:100) at room temperature fore 30 min in a wet chamber. Wash twice in PBS for at least 10 min each. Tertiary Antibody: Incubate with mouse monoclonal peroxidase anti-peroxidase (PAP) complex (diluted 1:100) for 30 min at room temperature in a wet chamber. Wash twice in PBS for at least 10 min each.
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Molecular Morphology in Human Tissues: Techniques and Applications Visualization: Immunopreciptates of PAP are visualized with diamidinobenzidine (DAB) and H2O2. Controls: For negative controls, either terminal transferase or BrdU were omitted in the reaction mixture. For positive controls, semi-thin sections from rat hypothalamus were used, which are normally devoid of apoptotic cells after DNA digestion with DNAse. The semi-thin sections were incubated at room temperature for 20 min with DNAse solution (Solution A) in DNAse buffer (Solution B). Solution A: DNAse solution: 200 ng DNAse dissolved in 1 ml DNAse buffer. Solution B: DNAse buffer: 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.1 mM CaCl2, 25 mM KCl
13.4.3.2 Reagents for Transglutaminase Immunohistochemistry DNAse II Tris buffer MgCl2 CaCl2 KCl Mouse Anti-BrdU antibody Mouse PAP Anti-mouse IgG DAB (3,3′-diamidinobenzidine) H2O2 (hydrogen peroxide) Normal goat serum
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Sigma D 8764 Sigma T 8280 Sigma M 1028 Sigma C7666 Sigma P9327 Progen 11200 Sigma P 2416 Santa Cruz Sc2039 Sigma 5905 Sigma H6520 Santa Cruz Sc2043
13.4.3.3
Suppliers
Progen: Maaβstrasse 30, 691123 Heidelberg; www.progen.de Santa Cruz Biotechnology: 2145 Delaware Avenue, Santa Cruz, CA 95060; www.scbt.com Sigma: www.sigma-aldrich.com 13.4. 4 Immunohistochemistry with Transglutaminase 13.4.4.1
Procedure
To stain the 0.5-µm thick sections, the Epon resin must be removed by treatment with 10% sodium methylate for 3 min, followed by a 1:1 mixture of methanol and benzene, for 2 min, and with acetone, 2× for 2 min each. After rinsing in 66 mM PBS, sections were stained for DNA fragmentation using terminal transferase and BrdU (see above). Primary antibody: Consecutive sections are incubated with goat anti-TTG polyclonal antibody. Immunogen for raising this antibody was purified guinea pig liver type II transglutaminase; the antibody recognizes type II transglutaminase, an 82kDa protein. Sections are incubated for 12 hr at 24°C with this primary antibody, diluted 1:1000 in PBS together with 1% rabbit normal serum. For controls, sections are incubated in normal goat serum applied in the same dilution but omitting the primary antibody. Secondary antibody: Sections were incubated for 60 min at 20°C with biotinylized rabbit anti-goat IgG antibodies (diluted 1:200; Sigma-Aldrich). Third layer: Avidin, labeled with horseradish peroxidase (HRP) (diluted 1:200; Sigma-Aldrich), for 60 min at 20°C.
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High-Resolution Visualization of Apoptotic Markers in Human Biopsies Visualization: Peroxidase was visualized with DAB (Sigma-Aldrich) and 0.1% H2O2 in PBS for 10 to 15 min at room temperature. Technical Hints: After each incubation with the antibodies, sections are washed twice in 66 mM PBS for 10 min at 20°C. After visualization, sections are dehydrated, cleared in xylene, and mounted with Entellan (Merck). Slides were photographed with a digital camera (PB 10, Olympus, Tokyo, Japan). 13.4.4.2
Reagents
Entellan Merck 107960 Avidin HRP Sigma 3151 Biotinylized antiGoat IgG Santa Cruz Sc 2774 Anti-Transglutaminase antibody Upstate 06-471 13.4.4.3
Suppliers
Santa Cruz Biotechnology: 2145 Delaware Avenue, Santa Cruz, CA 95060; www.scbt.com Upstate Biotechnology: 199 Saranac Avenue, Lake Placid, NY 12946; www.upstatebiotech.com Sigma: www.sigma-aldrich.com Merck: Frankfurter Str. 250, D-64243 Darmstadt; http://pb.merck.de
13.5 RESULTS Sections of human duodenum showed intense transglutaminase immunostaining, mainly at the top of the duodenal villi (see Figure 13.1A). Most of the enterocytes stained intensely for TTG, but only in the cytoplasm. Nuclei and mucous goblets were spared. When we
compared the sections stained for TTG with the following sections stained for DNA fragmentation, we could see clear correlation of TTG within cytoplasm and staining for DNA fragmentation in the nuclei (see arrows in Figures 13.1A and B). Cells with TTG immunoreactivity in the cytoplasm always had nuclei with strong DNA fragmentation. BrdU immunostaining was confined to the nuclei of enterocytes and some cells in the stroma of the villi. These cells are most likely phagocytes. In some cells showing DNA fragmentation, there was low or no staining for TTG (Figures 13.1A and B, aster). This holds true mainly for cells in the middle part of the villi. When using the same technique on semi-thin sections of human kidney biopsies containing epithelia with low cellular turnover, we found DNA fragmentation mainly in the epithelial cells of the distal tubules. Sections of the human kidney from a patient with nephrosclerosis showed intense TTG staining mainly in the distal tubules (Figure 13.2A, a and b). In few cases also in single cells of the proximal tubules. Cells showing immunostaining for TTG always had nuclei with strong DNA fragmentation, as can be seen in the following sections stained with BrdU-antibody (Figure 13.2B, a and b). Most of the proximal tubules were free of TTG staining and likewise without DNA fragmentation (Figure 13.2A at c, and Figure 13.2B at c). In some of the proximal tubules, single nuclei with strong DNA fragmentation were found (Figure 13.2B, arrow). In these cells, no TTG immunoreactivity could be seen (Figure 13.2A, arrow). Glomeruli in most cases showed neither TTG staining nor DNA fragmentation (Figures 13.2A and 13.2B, arrow). 205
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Figure 13.1 Semi-thin sections of human duodenum showing TTG staining (A) in many enterocytes and in the stroma of the villus tip. Arrows point to enterocytes with stong TTG staining in the cytoplasm. The asterisk marks an enterocyte with weak/no TTG staining. In the consecutive section stained for DNA-fragmentation (B), nuclei with strong DNA fragmentation can be seen in all enterocytes with strong TTG staining (two of them are marked with arrows), and some with DNA fragmentation but weak or no TTG staining (asterisk). The asterisk indicates the position of a cell, which is essentially unstained in A and B. (Original magnification ×1250)
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Figure 13.2 Semi-thin section of human kidney from a patient with nephrosclerosis. Distal tubules are clearly marked with strong TTG staining (A) in the cytoplasm of epithelial cells (for example, a and b), whereas in proximal tubules TTG staining is very low or absent (arrow in A). In the consecutive section (B), all cells stained for TTG have nuclei showing strong DNA fragmentation (for example, a and b). One cell in a proximal tubule with strong DNA fragmentation (arrow in B) has no TTG staining. (Original magnification ×1100)
13.6 DISCUSSION Serial sectioning of human biopsies with the semi-thin technique suggested here allows one to clearly correlate different markers on the single-cell level. In human duodenum, immunostaining with TTG always correlated with strong DNA fragmentation, an indication for apoptosis. We
also found some cells showing nuclei labeled by the ISEL technique, an indication of DNA strand breaks, but no TTG staining. These cell must be considered viable cells, without any signs of apoptosis. DNA fragmentation may occur in many more or less healthy cells due to activity of topoisomerase or oxidative damage, without leading to apoptosis [1]. 207
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Molecular Morphology in Human Tissues: Techniques and Applications The same results could be found in serial sections of the human kidney. A strict correlation between TTG staining and DNA fragmentation could be demonstrated. This indication of apoptosis was seen mainly in the distal tubules. Whether the massive destruction of cells in the distal tubules is linked to nephrosclerosis is not clear, because distal tubules in “normal” kidney biopsies usually also show many cells with DNA fragmentation [2]. It is, however, most likely that massive destruction of epithelial cells by apoptosis, without compensatory proliferation, may finally lead to loss of the distal tubules, loss of function, and the clinical manifestation of nephrosclerosis.
8.
9.
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References 1.
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Aschoff, A. and Jirikowski, G.F., Apoptosis: correlation of cytological changes with biochemical markers in hormone-dependent tissues, Hormon. Metab. Res., 29, 535, 1997. Aschoff, A. et al., Colocalization of BAX and BCL-2 in small intestine and kidney biopsies with different degrees of DNA-fragmentation, Cell Tiss. Res., 296, 351, 1999. Ansari, B. et al., In situ end labeling detects DNA strand breaks in apoptosis and other physiological and pathological states, J. Pathol., 170, 1, 1993. Gorczyka, W. et al., Detection of apoptosis associated DNA strand breaks in fine needle aspiration biopsies by in situ end labeling of fragmented DNA, Cytometry, 15, 169, 1994. Migheli, A. et al., A study of apoptosis in normal and pathologic nervous tissue after in situ end labeling of DNA strand breaks, J. Neuropathol. Exp. Neurol., 53, 606, 1994. Aschoff, A., Jantz, M.S., and Jirikowski, G.F., Enzymatic in situ tailing with bromodeoxyuridine: a novel technique for high resolution visualization of apoptosis, Hormon. Metabol. Res., 28, 311, 1996. Amendola, A. et al., HIV-1 gp120 dependent induction of apoptosis in antigen-specific human T cell clones is characterized by tissue transglutaminase expression and prevented by cyclosporin A, FEBS Lett., 339, 258, 1994.
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Bianchi, L. et al., Abnormal Bcl-2 and tissue transglutaminase expression in psoriatic skin, J. Invest. Dermatol., 103, 829, 1994. Cummings, M.C., Apoptosis of epithelial cells in vivo involves tissue transglutaminase upregulation, J. Pathol., 179, 288, 1996. Fesus, L., Transglutaminase-catalyzed protein cross-linking in the molecular program of apoptosis and its relationship to neuronal processes, Cell. Mol. Neurobiol., 18, 683, 1998. Johnson, G.V.W. et al., Transglutaminase activity is increased in Alzheimer’s disease brain, Brain Res., 751, 323, 1997. Piacentini, M. and Autuori, F., Immunohistological localization of tissue transglutaminase and Bcl-2 in rat uterine tissues during embryo implantation and post partum involution, Differentiation, 57, 51, 1994. Polakowska, R.R. et al., Apoptosis in human skin development: morphogenesis, periderm, and stem cells, Dev. Dyn., 199, 176, 1994. Melino, G. and Piacentini, M., Tissue transglutaminase in cell death: a downstream or a multifunctional upstream effector? FEBS Lett., 430, 59, 1998. Greenberg, C.S., Birckbichler, P.J., and Rice, R.H., Transglutaminases: multifunctional cross linking enzymes that stabilize tissues, FASEB J., 5, 3071, 1991. Hilton, D.A., Love, S., and Barber, R., Increased endothelial expression of transglutaminase in glioblastomas, Neuropath. Appl. Neurobiol., 23, 507, 1997. Aschoff, A.P., Günther, E., and Jirikowski, G.F., Tissue transglutaminase in the small intestine of the mouse as a marker for apoptotic cells. Colocalization with DNA-fragmentation, Histochem. Cell. Biol., 113, 313, 2000. Lotze, U. et al., Damaged myocytes as detected by colocalization of DNA-fragmentation and tissue transglutaminase. Prognostic significance in enterovirus-associated dilated cardiomyopathy, Eur. J. Clin. Invest., 31, 744, 2001. Romeis, B., Mikroskopische Technik. Oldenbourg, München, 1948. Jirikowski, G.F., Ramalho-Ortigao, J.F., and Caldwell, J.D., Transitory coexistence of oxytocin and vasopressin in the hypothalamo neurohypophysial system of parturient rats, Hormon. Metabol. Res., 23, 476, 1991.
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Three-Dimensional Full Color Demonstration of Bright-Field and Fluorescence Microscopic Preparations: The Digital Optical Microscope Gerhard W. Hacker, Veit Schubert, Leo Wollweber, Michael Schwertner, and Dietmar Schwertner
14.1 INTRODUCTION When examining a microscopic preparation, the histologist or molecular morphologist usually looks at a two dimensionally (2D) imaged three-dimensional (3D) structure from which she or he may be able to approximate the real 3D structure. There are, however, situations in which it would be especially helpful to actually look at a 3D image directly: The spatial structure of an unknown microscopic specimen cannot be understood by a 2D image itself, because it requires the human imagination based on previous experience. Even 2D extended focus imaging does not improve this situation. In many areas of microscopy, a clear understanding of microscopic structures can be only appreciated by a real 3D image, allowing a genuine space perception. For molecular morphologists, a number of areas exist in which observation of the 3D structure would add valuable additional insights, for example, when neurobiologists attempt to trace neuropeptides/ 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
nerve fibers and their connections within tissue sections or whole mount preparations, when the 3D intracellular distribution of cytosolic antigenic structures is of interest, and, when the simultaneous visualization of the 3D intranuclear distribution for several gene loci is required. Different technical solutions have been developed to produce 3D images of a higher magnification than 200 times, the practical maximum of a conventional stereo microscope. Such solutions include the “space image microscope” (Zeiss), the “confocal laser scanning microscope (CLSM)” (Biorad, Leica, Nikon, Olympus, Zeiss), and the “restauration microscope” systems based on deconvolution methods (Leica). However, a high-resolution 3D image in true-color using the trans-illumination technique, as well as 3D time lapse sequences, cannot be produced by such systems, due to their construction design [1, 2].
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Molecular Morphology in Human Tissues: Techniques and Applications To overcome such limitations, digitaloptics (Jena, Germany) has developed a “digital optical microscope” based on a new 3D reconstruction method. The digital optical microscope (DOM) is capable of generating spatial images of very high resolution using the trans-illumination technique, in combination with optical contrast techniques such as differential interference contrast (DIC), or polarization contrast. For the first time, the DOM technique allows visualizing spatial structures even without applying fluorescence-based preparations [3–5]. This new quality is represented by a complete true-color 3D image, which can be achieved by visualizing conventional histological, cytological, and molecular morphological preparations. This chapter provides an overview of the description of this technology and shows examples from human tissue sections as well as from other fields of biology. A number of possible applications of this type of stereoscopic imaging are discussed, and the pertinent literature is reviewed. For the molecular morphologist, examples are shown that illustrate how 3D DOM can add further aid in understanding the structures detected. 3D images were obtained from gold- and silver-staining methods applied for immunohistochemistry (immunogold-silver staining, IGSS) [6–19] and for the demonstration of specific nucleic acid sequences [12, 20–27]. These applications show a fascinating merging of two worlds: the specific demonstration of the 3D cellular or extracellular distribution of an antigen (such as a protein or a neuropeptide) or a given nucleic acid sequence, together with the visualization of the underlying tissue structure stained with conventional histochemical counterstains, in full color. All this is possible in nonbleaching, permanent preparations for light microscopy (LM).
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14.2 METHODOLOGY 14.2.1 General Features To create high-resolution 3D images, the DOM uses image processing combined with nonlinear algorithms [28]. The term ”digital optical microscope“ has been selected because the system combines optical bright-field imaging with digital image processing. As a result, this leads to a new image quality that could not be achieved by pure optical means. The DOM cannot only produce bright-field and fluorescent 3D images, but also has a number of additional features. All conventional optical contrasting techniques commonly available in standard LM can be applied. Epifluorescence, DIC, and trans-illumination or epi-illumination polarization can be performed. The application and/or combination of such different contrast techniques together with nondestructive specimen preparation also allows in vivo observations as well as real in vivo 3D-3D co-localizations without major artifacts. Classical histochemical staining techniques can be used and could give rise to a renaissance in the field of structural imaging. For more information on histotechnology, see the following Web sites: http://members.pgonline.com/~bryand/ StainsFile/ and http://www.nottingham. ac.uk/pathology/pathprot.html. In addition to optical contrast, computer-generated contrasts such as dark-field simulation allow the creation of 2D (extended focus) and 3D images of improved contrast. The system thereby does not reduce the resolution of the microscope and the visualization of the full potential specimen depth is possible. One advantage of the DOM compared to the CLSM is that a conventional LM is used, providing high photon transfer efficiency. Combined with highly sensitive
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Bright-Field and Fluorescence Microscopic Preparations cameras, this is the prerequisite that allows for minimizing the light energy impact on the specimen [29]. Therefore, this new 3D reconstruction technique also enables the user to study fluorescence-labeled preparations during a relatively long time (depending on the dye used), as photo-bleaching can be reduced significantly. The combination of digital structure with information-driven imaging offers the possibility to combine two or more 3D images of the same specimen position produced by different contrast techniques. This enables a real 3D-3D co-localization of specimen features. A possible application of this feature is the simultaneous demonstration of several gene sequences by multicolor fluorescence in situ hybridization (FISH). The DOM system is completely automated and can deliver a 3D image within less than 20 sec. Consequent-
ly, it also allows producing time-lapse sequences (“4D microscopy”) to observe slowly moving processes in living cells or organisms. 14.2.2 The DOM System The system is based on a conventional bright-field LM with telecentric imaging — which is the case in nearly all modern LMs. Microscopes containing an electronic focus movement and a serial computer interface, as evidenced, for example, by the Axioplan 2 (Zeiss), DMRX (Leica Microsystems, Wetzlar, Germany), Eclipse 1000 (Nikon Corporation, Tokyo, Japan), and Provis AX 70 (Olympus, Tokyo, Japan), are especially well suited. Older LM types can be also used, but it is necessary to integrate piezoelectric drivers into the microscope stage as is possible for upright but also inverted microscopes (Figure 14.1).
Figure 14.1 Double DOM at IPK Gatersleben: Two Zeiss microscopes (Axiovert 100TV and Axiophot) equipped with piezoelectrically driven stages are integrated into one DOM system. Both microscopes can be steered separately. A highresolution color TV camera and a black/white camera can be used reciprocally. Shutter glasses allow one to observe the 3D images directly at the computer screen.
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Molecular Morphology in Human Tissues: Techniques and Applications The highly complex integrated software program acts in such a way that there are only small differences in managing the DOM compared to a conventional LM. The primary advantages of the DOM are that (1) one can watch the images in full color with high sensitivity and resolution, and (2) the produced 3D image only combines in-focus information in every area from the section or cytological preparation. The software calculates and excludes other picture data derived from out-of-focus tissue portions on the basis of sharpness criteria. The images gained are therefore of outstanding sharpness and clarity. The principle applied by the DOM system to produce a 3D image is shown in Figure 14.2. It allows for fully automated microscope control and data transfer to the computer, as well as the automatic analysis of image stacks, the reconstruction of truecolor high-resolution stereoscopic images of the specimen, and the 3D visualization of the image. For display purposes, the 3D images can be presented as stereo images at the computer monitor using LCD stereo shutter glasses, or by simulated rotation of the extended focus image. Viewing the LCDshuttered monitor picture allows for far more exact monitoring than possible with prints. However, to publish 3D images in
journals or books, it is necessary to print them on paper. This can be done by printing stereo pairs on photographic paper, or by converting the data into red-green anaglyph images, such as those presented in this chapter (Figures 14.3, 14.4, 14.5, and 14.6). Printed 3D images require special red-green glasses (for anaglyph images), or, in the case of printing full-color stereo pairs of pictures for the left and right eye, prism glasses for the viewer, as commonly used in 3D imaging in microscopy. With some experience, stereo pairs can also be observed without glasses from a distance of about 30 cm (about 10 inches): the observer must then look at the stereo pair by accommodating her or his eyes at “distance”; the pair then should “melt into one,” and then the observer’s brain produces the impression of a spatial image. 14.2.3 Commercial Sources DOM systems are available from digitaloptics: Closewitzerstr. 3, D-07743 Jena, Germany; email:
[email protected]; website: http://www.digitaloptics.de. Prismatic eyeglasses to watch printed 3D stereo pairs in black and white or in full color can be purchased from Reel 3-D Enterprises, Inc.: P.O. Box 2368, Culver City, CA 90231 and
Figure 14.2 Principle for production of a 3D image by the DOM system.
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Figure 14.3 (Color Figure 14.3 follows page 106.) Delicate basket cell nerve fibers in cerebellum, partly in close contact with a Purkinje cell (located in the middle of these photomicrographs), demonstrated with indirect IGSS using 5-nm colloidal gold particles adsorbed to second-layer goat antibodies against IgG of rat. Silver acetate AMG was used for gold amplification [8]. As primary reagent, a rat monoclonal antibody against neurofilament proteins was applied [7]. In this preparation, 3D brings an enormous amount of additional information on the spatial composition of the nerve fiber network. A and B are a stereo pair, showing the preparation in true color. Prism eyeglasses must be used for observing. C and D are anaglyph images of the same preparation, and must be observed with red-green eyeglasses to obtain the 3D effect: C is obtained from conventional bright-field LM with the DOM, and D is an electronically simulated dark-field image generated by the DOM. IGSS is seen in black; counterstaining was performed with eosin and Nuclear Fast Red.
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Figure 14.4 (Color Figure 14.4 follows page 106.) 3D demonstration of single muscle cells with monoclonal mouse antibodies against desmin [10], stained with indirect IGSS, 5-nm colloidal gold and silver acetate AMG, as in Figure 14.3. Muscle striation can be observed in highest resolution, and it is also possible to recognize hexameric structures in 3D — which would not be possible using a conventional two-dimensional setup. IGSS in black color; counterstained with hematoxilin. A and B are a stereo pair in full color, to be observed using prism eyeglasses. C and D are anaglyphic images (medium-high and very high power magnifications), and red-green glasses should be used for observation.
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Figure 14.5 (Color Figure 14.5 follows page 106.) 3D images of lymph node micrometastasis derived from an ileal VIPoma, a neuroendocrine tumor producing vasoactive intestinal polypeptide (VIP). Indirect IGSS with 5-nm colloidal gold, silver acetate AMG and primary antibodies against VIP were used. All three images must be observed using redgreen glasses. A and B are images from bright-field LM obtained with different settings of the microscope condensor iris and contrasting, and C is from computer-simulated dark field. All three images allow recognizing the typical appearance of a cut “solid” structure (every tumor cell is immunoreactive to VIP), thus giving the impression of a polygon-shaped wheel, embedded in loosely connected lymphatic cells.
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Figure 14.6 (Color Figure 14.6 follows page 106.) Super-sensitive in situ hybridization with single copy efficiency, performed to detect human papillomavirus (HPV) 16/18 in condyloma acuminatum. A biotinylated genomic DNA probe to HPV 16/18 (Enzo, New York, NY) was used, and a CARD-Nanogold-silver procedure was applied [12–17, 25–27]. Using biotinylated tyramides, as patented by Perkin Elmer and licensed to DakoCytomation (“Genpoint” kit, DakoCytomation, Glostrup, Denmark) together with Nanogold™-labeled streptavidin (Nanoprobes, Inc., Yaphank, NY) and silver or gold enhancement, single copy sensitivity can be obtained (see also Chapter 6). However, by 3D analysis, we can observe that this procedure can destroy morphology to a certain degree. It can be seen at first sight that some of the HPV-16/18 containing and nuclei have been loosened and conveyed to slightly changed locations — some of them now “hanging” on the glass slide. Of course, one might already assume that procedures in which heating/temperature changes, proteinase treatment, and long-lasting washes are to be used might cause artifacts — these can be readily demonstrated using the 3D DOM. Both images shown here are anaglyphic and need to be observed with red-green glasses. A is a bright-fieldobtained image without further computerized contrasting, whereas B represents a computer simulated dark-field image. Counterstaining with Nuclear Fast Red; 100X objective.
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Bright-Field and Fluorescence Microscopic Preparations sold under the term “plastic handheld 3-D print viewer,” Article No. 2018. They are well suited for 3 × 3inch stereo pairs, as in some of the figures in the present chapter. Redgreen anaglyphic eyeglasses to watch red-green 3D images, as also shown in some figures in this chapter, can be purchased from the same company (e.g., Article No. 7021); Web site: http://www.stereoscopy.com/reel3d.
In general, for investigating 3D structural relationships, it is sometimes necessary to customize the preparation methods for the intended purpose. When preparations such as histological sections or wholemount preparations are being prepared for 3D examinations, one should avoid any treatment that could destroy or substantially change the spatial morphological structure. It is also wise to cut thicker sections than usual, in order to gain more information in this approach.
14.2.4 Materials and Methods The DOM can document practically every kind of histological or cytological preparation spatially. These include histologic sections from about 3 to 20 µm thickness, whole-mount preparations of up to 80 µm thickness, cytological preparations using diachromes or fluorescent dyes, and living specimens embedded into coverslip chambers. Nearly all known special techniques of contrast amplification or of false-contrasts can be applied, if the LM used is equipped for it. In particular, native structures should be maintained without artifacts; but in biological specimens, the preparation process may cause some degree of distortion of the spatial relationships. Special care should therefore be taken to use such fixation and preparation methods minimizing morphological artifacts. The ability to produce 3D images by applying nondestructive contrast techniques such as DIC or polarization is of special value to drastically reduce the occurrence of artifacts. The potential of DOM to generate a 3D true-color image within a short time allows the monitoring of dynamic processes in living cells or the observation of the spatial distribution of markers. To carry out the important step from a static to a dynamic image of the cell, such nondestructive 3D cell monitoring is becoming of increasing interest [30, 31].
14.3 APPLICATIONS AND DISCUSSION The possibility of analyzing the specimen in its spatial reality represents one of the major achievements of modern structural research. The advantages of the DOM described above should lead to execution of the important step to spatial structural analysis in various areas of medical, biological, and nonbiological research. Numerous potential applications exist in material research, zoology, botany, and taxonomy. This chapter discusses some applications in immunohistochemistry and molecular morphology, and also demonstrates examples from certain fields of general biology in which insights into complex structure-function relationships mediated by 3D analyses are of common interest. 14.3.1 Applications in Immunohistochemistry To demonstrate immunostained structures contained in tissue sections or cells by 3D microscopy, the use of a particulate labeling system offers distinct advantages. Using immunogold-silver or immunogoldgold staining (IGSS or IGGS, respectively), in which either tiny particles of colloidal gold (1 to 5 nm in diameter, e.g., from Aurion, Wageningen, the Netherlands) or 217
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Molecular Morphology in Human Tissues: Techniques and Applications gold clusters (Nanogold, gold core diameter 1.4 nm; Nanoprobes, Inc., Yaphank, NY) are the labels adsorbed or covalently bound to antibodies or other macromolecules (such as streptavidin), permits higher resolution than that possible with enzymatic or fluorescent labels [6–19]. An example from neurobiology is shown in Figure 14.3; in this field, it is sometimes necessary to follow the 3D lineage of networks of central or terminal nerve fibers [7]. The third dimension adds an improved comprehension of the complexity of neuronal structures, and a better understanding of axonal/dendritic/synaptic connections to other cells or structures, such as to pericarya or to muscle fiber endplates. Numerous applications — for example, in brain research, or in the investigation of the peripheral nerve system (e.g., terminal nerve fiber distribution in the skin) — are possible. Indirect IGSS with silver acetate autometallographic development has been applied [6, 8, 10, 12], and the resolution and the spatial 3D images obtained with high magnification objectives (100×) in the LM are remarkable.
the 3D structure of muscle striation is demonstrated with antibodies to desmin and indirect colloidal gold IGSS [10]. The striation is visualized with remarkably clear detail; and although the morphology is somewhat compromised by the embedding/sectioning/staining processes, it is even possible to visualize the hexameric structure focally. Figure 14.5 shows an example of a neuroendocrine tumor whose main neuropeptide is demonstrated by IGSS [32]. This case of a VIPoma micrometastasis shows the typical 3D plate-like structure given in histological sections when a mass of tumor cells contains high amounts of the cytosolic substance to be detected. 14.3.2 Applications in Molecular Morphology
Other approaches, using double or triple immunofluorescence, may allow the simultaneous demonstration of 3D images from nerve fibers containing different neuropeptides or other neuronal markers with relative ease, for example, labeling with fluorescein isothiocyanate (FITC) (green fluorescence), rhodamine isothiocyanate (RITC) or Texas red (red fluorescence), and AMCA (blue fluorescence), or with the newer generation of fluorescent dyes such as the cyanines (e.g., Cy-3) and Alexa dyes yielding enhanced durability of the preparations. (See also Chapters 1 and 2 of this book.)
Figure 14.6 demonstrates examples obtained from in situ hybridization (ISH) methods. Using Nanogold-silver ISH, a preparation of condyloma acuminatum is shown, hybridized with a biotinylated genomic DNA probe to human papillomavirus (HPV) types 6/11 (Enzo, New York) and the staining method described in [12, 20–27]. One can see that in conventionally processed (formalin-fixed, paraffin-embedded) and then heated (for the hybridization) tissue, together with proteinase treatment and the numerous washing steps, the 3D morphology may be severely affected, and the image obtained (although this is not so well seen in a normal (2D) LM) shows hybridized and partly pycnotic nuclei that have, in part, “moved” from their original places and are “condensed.” The image shown here has been obtained with computerized darkfield simulation.
Another application of immunohistochemistry is shown in Figure 14.4. Here,
Analysis of the spatial chromatin distribution within interphase nuclei is an impor-
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Bright-Field and Fluorescence Microscopic Preparations tant topic of recent basic biology research. It is one of the prerequisites to understand how DNA sequences are expressed differentially during the cell cycle and within different tissues. Figure 14.7 shows a 3D image of FISH, using Alexa 488 to demonstrate a specific DNA sequence: In this example, the elegance of the 3D DOM principle permitting visualization of spatial chromatin loops, is demonstrated. 14.3.3 Applications in General Biological Research A variety of applications of DOM technology in general biological research have been already explored. As an example from immunofluorescence, parasite-infected cells are shown in Figure 14.8. A FITC-labeled antibody against secretory parasite antigens allows one to visualize the spatial distribution of secretory parasite antigens within these infected cells. Recently, there has been increasing interest, particularly in the field of live cell imaging in basic research. Various biological objects can be directly analyzed in vivo using bright-field contrast techniques such as DIC. However, the spatial extension of such a living specimen requires acquisition of image stacks to produce images showing the 3D reality. This task can be performed for the first time by the DOM, very rapidly and with a superior image quality, which is also the prerequisite for production of 3D time-lapse sequences. Somatic metaphase chromosomes stained with Hoechst 33342 and 3D-3D co-localized in a living kangaroo rat cell, acquired in DIC, are demonstrated in Figure 14.9. An additional feature is the ability to optically open objects, as demonstrated in Figure 14.10. In contrast to the closed cell of a Tradescantia stamen hair, the optically
opened cell shows the internal organelles with high contrast. Recent advances in life cell research were also achieved by applying Green Fluorescent Proteins (GFP) and its variants. Bereczky et al. [33] successfully used the DOM to identify transiently expressed tomato metal transporter genes fused with GFP in living yeast cells. Figure 14.11 is an example of a real 3D-3D co-localization of nuclei labeled with histone H2B-Yellow Fluorescent Protein within a living Arabidopsis root. As is visible in this picture pair, computer-simulated dark-field images produced by the DOM and 3D-3D merged with fluorescence images can clearly enhance the contrast. 14.5 THE FUTURE 3D imaging in full color can be understood as an interesting addition to the huge reservoir of methodologies already available. However, it may become much more: DOM and related technologies show a remarkable potential to initialize a new era of spatial imaging research. The range of advantages already discussed here, as well as the manifold new applications not yet foreseeable, may lead to a variety of hitherto unclarified insights into the structure and structural-functional relationships of molecular complexes in intra- and extra-cellular structures. Combining fullcolor 3D imaging with the high specificity of immunocyto/histochemistry and molecular morphology will add the “further dimension” necessary for a better understanding of medicine and biology. It may even be possible that a new discipline could be born: “3D imaging molecular morphology.” As soon as “normal” spatial structures of certain proteins, peptides or nucleic acid sequences, or specific lectinbinding sites (detected by gold-silver based 219
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7A
7B
8A
8B
9A
9B
Figures 14.7–14.9 (Color Figures 14.7–14.9 follow page 106.) [7] FISH of a Alexa 488 labeled 66,131 bp DNA sequence from chromosome 1 of Arabidopsis thaliana to a 2C nucleus isolated from root tissue: Two spatial chromatin loops originating from each of the two homologs are clearly visible. The nucleus was counterstained with 4′,6-diamidino-2phenylindole (DAPI). [8] MDBK (Madin Darbin Bovine Kidney) cells infected with Eimeria tenella (Coccidia, Apicomplexa, Protozoa). These parasites cause severe disease in chicken (coccidiosis). Parasite development in cell culture has been stopped by fixation with paraformaldehyde. After permeabilization of cell membranes with cold acetone, the binding sites of a monoclonal antibody against secretory parasite antigens were visualized with anti-mouse FITC. The green fluorescent signal shows parasite antigens secreted within an infected host cell. (Preparation by A.M. Noutossi and R. Entzeroth, Technical University of Dresden, Germany.) [9] DOM image of an adherent live epithelial-like kangaroo rat (PtK2) cell grown on a glass slide and stained with Hoechst 33342, which binds AT pairs in the DNA and is able to enter viable cells without the need for fixation. Chromosomes in the equatorial plate exhibit bright blue fluorescent staining. The image shown was obtained by overlaying the fluorescent image with the differential interference contrast (DIC) image. Objective: 100X. These three stereo pairs should be observed with prism glasses in color.
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Figures 14.10–14.11 (Color Figures 14.10–14.11 follow page 106.) [10] “Optical surgery” of a living Tradescantia stamen hair cell in true-color DIC compared to a “closed” cell (top); the 3D structure of the nucleus inside connected with the cell wall by cytoplasm strands is clearly visible. [11] Real 3D-3D “co-localization” in living plant tissue: the distribution of nuclei expressing a histone H2B-YFP construct is visible within an Arabidopsis thaliana root acquired in DIC but shown in DOM dark-field simulation to improve contrast. Plants were grown on agar medium in coverslip chambers under sterile conditions. These two stereo pairs should be observed with prism glasses in color.
lectin histochemistry) have been mapped, experiments in cell culture may identify particular alterations characteristic for the approach utilized; this may lead, for example, to a new dimension of understanding drug interference. Specific 3D-3D colocalizations and 4D life cell imaging, as well as a rejuvenation of classical histochemistry that — in the case of gold-silver or gold-gold labeling — can even be applied as a multicolor counterstain, could be among the most promising new techniques of biomedical research.
References 1.
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Schubert, V., Räumliche Abbildungen mit konventionellen Lichtmikroskopen, GIT LaborFachzeitschrift, 44(8), 942, 2000. Schwertner, M., Schwertner, D., and Schubert, V., Digitaloptische 3D Mikroskopie — eine neue Entwicklung im Vergleich, Transcript. Laborwelt, 2, 15, 2002. Schwertner, M., Schwertner, D., and Schubert, V., 3D imaging with the Digital Optical Microscope, G.I.T. Imaging & Microscopy, 2, 48, 2000. Schubert, V., Schwertner, M., and Schwertner, D., Das Digitaloptische Mikroskop — Ein neues System zur Erzeugung räumlicher Abbildungen in der Lichtmikroskopie, Naturwiss. Rdsch., 54, 637, 2001.
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Schubert, V., Schwertner, M., and Schwertner, D., Das Digitaloptische Mikroskop - Wichtige Innovation zur dreidimensionalen Mikroskopie, Mikrokosmos, 91, 261, 2002. Hacker, G. W. et al., The immunogold-silver staining method. A powerful tool in histopathology, Virchows Arch. A Pathol. Anat. Histopathol., 406, 449, 1985. Hacker, G.W. et al., Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs, Histochemistry, 82, 581, 1985. Hacker, G.W. et al., Silver acetate autometallography: an alternative enhancement technique for immunogold-silver staining (IGSS) and silver amplification of gold, silver, mercury, and zinc in tissues, J. Histotechnol., 11, 213, 1988. Hacker, G.W. et al., The use of silver acetate autometallography in the detection of catalytic tissue metals and colloidal gold particles bound to macromolecules, Prog. Histochem. Cytochem., 23, 286, 1991. Hacker, G.W. and Danscher, G., Recent advances in immunogold-silver staining-autometallography, Cell Vision, 1, 102, 1994. Hacker, G.W. et al., Electron microscopical autometallography: immunogold-silver staining (IGSS) and heavy-metal histochemistry, Methods, 10, 257, 1996. Hacker, G.W. and Gu, J., Gold and Silver Staining: Techniques in Molecular Morphology, CRC Press, Boca Raton, FL, 2002. Hainfeld, J.F., A small gold-conjugated antibody label: improved resolution for electron microscopy, Science, 236, 450, 1987. Hainfeld, J.F., Gold cluster-labelled antibodies, Nature, 333, 281, 1988. Hainfeld, J.F. and Furuya, F.R., A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177, 1992. Hainfeld, J.F., Labeling with Nanogold and Undecagold: techniques and results, Scanning Microsc. Suppl., 10, 309 and 322, 1996. Hainfeld, J.F. and Powell, R.D., New frontiers in gold labeling, J. Histochem. Cytochem., 48, 471, 2000.
18. Holgate, C.S. et al., Surface membrane staining of immunoglobulins in paraffin sections of nonHodgkin’s lymphomas using immunogold-silver staining technique, J. Clin. Pathol., 36, 742, 1983. 19. Holgate, C.S. et al., Immunogold-silver staining: new method of immunostaining with enhanced sensitivity, J. Histochem. Cytochem., 31, 938, 1983. 20. Cheung, A.L. et al., Detection of human papillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization, Mod. Pathol., 12, 689, 1999. 21. Graf, A.H. et al., Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma, Appl. Immunohistochem. Mol. Morphol., 8, 300, 2000. 22. Hacker, G.W., High-performance Nanogold-silver in situ hybridisation, Eur. J. Histochem., 42, 111, 1988. 23. Hacker, G.W. et al., Application of silver acetate autometallography and gold-silver staining methods for in situ DNA hybridization, Chin. Med. J. (Engl.), 106, 83, 1993. 24. Hacker, G.W. et al., High-performance Nanogold in situ hybridization and in situ PCR., Cell Vision, 3, 209, 1996. 25. Hacker, G.W. et al., In situ localization of DNA and RNA sequences: super-sensitive in situ hybridization using streptavidin-Nanogold-silver staining: minireview, protocols, and possible applications, Cell Vision, 4, 54, 1997. 26. Hacker, G.W., High performance Nanogold-silver in situ hybridisation, Eur. J. Histochem., 42, 111, 1998. 27. Zehbe, I. et al., Sensitive in situ hybridization with catalyzed reporter deposition, streptavidinNanogold, and silver acetate autometallography: detection of single-copy human papillomavirus, Am. J. Pathol., 150, 1553, 1997. 28. Schwertner, D. and Schwertner, M., 1995, Verfahren zur hochauflösenden Stereomikroskopie. Patent DE 19504108C2. 29. Sandison, D.R. et al., Quantitative fluorescence confocal laser scanning microscopy (CLSM), in Handbook of Confocal Microscopy, Pawley, J.B., Ed., Plenum Press, New York, 1995.
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Bright-Field and Fluorescence Microscopic Preparations 30. Rietdorf, J. and Zimmermann, T., 4D microscopy: exploring time and space, G.I.T. Imaging & Microscopy, 2, 44, 2000. 31. Keller, P., Fluorescence microscopy in living cells, Bioforum Int., 4, 104, 2000. 32. Graf, A. et al., Archival gastrointestinal carcinoids evaluated by immunogold-silver staining (IGSS) and histochemical silver staining techniques, Cell Vision, 3, 40, 1996.
33. Bereczky, Z. et al., Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato, J. Biol. Chem., 278, 24697, 2003.
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Visualization of Signal Transduction Pathways in Real Time: Protein Kinase C and Phagocytosis Michelle R. Lennartz, Pamela M. Brannock, and Joseph E. Mazurkiewicz
15.1 INTRODUCTION The ability to visualize signal transduction pathways in real time has resulted from the confluence of powerful analytical tools from molecular biology and confocal microscopy. In particular, monitoring green fluorescence protein (GFP)-conjugated Protein Kinase C (PKC) isoforms that were expressed in cells in culture has permitted the investigation of PKC movement in living macrophages as they undergo immunoglobulin-G (IgG) mediated phagocytosis of either pathogens or model targets (IgG-coated glass beads). Inactive PKCs are localized in the cytosol, and following activation, they translocate to the plasma membrane or intracellular organelles. In the case of phagocytosis, one isoform, PKC-ε, specifically translocates to the site of the forming phagocytic cup, remains associated with the membrane of the forming phagosome, returning to the cytosol upon complete engulfment of the target. The ability to manipulate DNA and introduce it into cells by transfection gave rise to several disciplines of molecular biology that rapidly evolved from a scientific 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
discipline into the versatile technique it is today. Early studies were done in Chinese hamster ovary (CHO) cells or COS-1 fibroblasts due to their high transfection efficiencies. However, the necessity to study cell type-specific signaling has increased the demand for more effective transfection reagents and led to the use of viruses as DNA delivery systems for cells that are difficult to transfect. Cells of the hematopoietic lineage, in particular, have been relatively refractory to these approaches, with low transfection efficiencies in mouse macrophages and few reports of transfection of human cells [1, 2]. To address this difficulty, this chapter introduces protocols that detail the construction of lentiviral vectors and their use in the transduction of human monocytic cells. Phagocytosis is the process by which specialized cells internalize particulate matter in a series of well-coordinated events. Peripheral blood monocytes or their differentiated counterparts, macrophages, are most commonly used for studies of this process, with both mouse and human cell
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Molecular Morphology in Human Tissues: Techniques and Applications lines being used for in vitro studies. Phagocytosis is ideal for visualizing signaling events because it is a localized process in which one particle (target) is engulfed within a single phagosome. Signaling molecules that contribute to uptake or that are activated upon receptor ligation translocate and concentrate beneath bound targets. Conversely, inhibition of upstream events blocks localization of subsequent signaling molecules. Several excellent reviews are available to those interested in the specifics of signal transduction during phagocytosis [3, 4]. This chapter presents protocols for studying the translocation of signaling molecules during IgG-mediated phagocytosis. Mouse macrophage cell lines are more readily transfectable than their human counterparts. Thus, protocols and procedures were developed first using such cells, and are presented as models to be used when human macrophages are studied following viral transductions. The chapter is divided into two parts: the first describes transient transfection of a mouse macrophage cell line and the second describes lentiviral transduction of the human monocytic cell line Mono Mac-6. For construction of expression plasmids, the reader is directed to molecular biology manuals (e.g., www.molecularcloning.com).
describe (1) the generation of the fluorescently labeled targets (sheep red blood cells and glass beads), (2) transient transfection of cells, (3) the imaging setup, and (4) the analyses of the data for the mouse macrophage model system. This is followed by a description of the development of the lentivirus delivery system presented in a series of protocols that will permit readers to adapt them to their own particular system. This second set of protocols includes production of the viral stock, transduction of human and mouse cells, and the characterization of the transduced cells by dual parameter flow cytometry. Characterization includes assays that examine (1) the phagocytic process itself and (2) the production of tumor necrosis factor-α (TNF-α), both of which demonstrate that the viral transduction process per se does not alter functions intrinsic to these cells. 15.3 PROTOCOLS FOR THE PREPARATION OF TARGETS The choice of fluorescent target depends on the assay being performed. In the studies described in this chapter, Alexa 568labeled IgG-opsonized glass beads were the targets used for analysis by confocal microscopy. Phycoerythrin-labeled sheep red blood cells (PE-SRBC) were used for dual parameter flow cytometry.
15.2 GENERAL COMMENTS The basic visualization experiments involve feeding Alexa 568 (Red) labeled IgG-opsonized targets to GFP-expressing macrophages and monitoring phagocytosis in real time by capturing dual-color confocal images of the fluorescence emitted by the GFP and the Alexa 568. The versatility of the technique lies in the fact that any GFP-conjugated signaling molecule can be transfected and imaged. The protocols will
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15.3.1 Protocol A: Preparation of IgGOpsonized Glass Beads Production of opsonized-fluorescent glass beads is a three-step process: 1. Generation of the Alexa 568 bovine serum albumin (BSA) 2. Coating the beads with Alexa 568BSA
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Visualization of Signal Transduction Pathways in Real Time 3. Opsonization of the coated beads with anti-BSA IgG This protocol uses 50 mg of 2-mm borosilicate beads. Once prepared, coated beads are good for about 1 week. All processing is done in 1.5-ml microfuge tubes to facilitate rapid washing. Beads are pelleted by a brief pulse in the microfuge. Remove the wash solution and vortex before adding the next solution. The final solution should have a homogeneous milky appearance, not particulate. Should aggregation occur, a brief (2 sec) sonication often breaks up the clumps. Unless otherwise noted, all steps can be done at room temperature. 15.3.1.1
15.3.1.2 1.
2.
3.
Materials and Reagents
Alexa 568 conjugation kit (Cat. No. A10238, Molecular Probes, Inc., Eugene, OR; http://www.probes.com/) Borosilicate glass beads (Cat. No. 9002, Duke Scientific, Palo Alto, CA; http://www.dukescientific.com/) Poly-L-Lysine (Cat. No. P1274, SigmaAldrich, Inc., St. Louis, MO; http:// sigma-aldrich.com/) DMP (dimethyl pimelimidate, Cat. No. 21667, Pierce Biotechnology, Rockford, IL; http://www.piercenet.com/) IgG-free bovine serum albumin (BSA; Cat. No. A-0281, Sigma-Aldrich, Inc.) Anti-BSA IgG (#55275, ICN Biochemicals/Cappel, Aurora, OH; http://www. icnbiomed.com/) Phosphate buffered saline (PBS): magnesium and calcium free, Dulbecco’s Phosphate Buffer Solution (Cat. No. 20-031-CV; MediaTech, Herndon, VA; http://www.cellgro.com/)
4.
5.
6.
7.
Labeling Procedure
Conjugate Alexa 568 to IgG-free BSA according to the manufacturer’s directions (Molecular Probes Alexa 568 conjugation kit). Acid-clean the glass beads by overnight soaking in 12 N HCl. As beads are stable in HCl, they can be stored indefinitely. Wash three times with 1 ml dH2O, and add 1 ml KOH. Fresh KOH is made each time by dissolving three pellets of KOH in 5 ml of 95% EtOH. Mix by inversion until beads are clotted and then mix for an additional 2 min. Wash three times with 1 ml dH2O and resuspend beads in 1 ml PBS. The cleaned glass beads are now ready for coating. To the beads, add poly L-lysine (PLL) to a final concentration of 50 µg/ml (5 µl of a 10 mg PLL/ml PBS stock in 1 ml of beads). Mix by inversion for 30 min. Wash three times with 1 ml PBS. Resuspend beads in 10 mM DMP (dimethyl pimelimidate) in PBS. Mix by inversion for 30 min. Wash two times with 1 ml PBS. Resuspend beads in 1 ml of 1% IgGfree BSA and then add 50 µl Alexa 568BSA. The volume of the fluorescent BSA solution may have to be adjusted, depending on the efficiency of conjugation in Step 1. Mix by inversion overnight at 4°C. Overnight incubation (>12 hr) is necessary to completely coat the beads. Incubation for less time results in beads that are nonspecifically taken up by macrophages. Incubation at 4°C minimizes bacterial growth. Wash two times with 1 ml of 0.5 M Tris, pH 8.0. Block residual reactive groups by incubation in 0.5 M Tris,
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Molecular Morphology in Human Tissues: Techniques and Applications pH 8, for 1 hr and then wash two times with 1 ml PBS. 8. These BSA beads (BBSA) are poorly phagocytic and serve as control particles for studying IgG-dependent signaling events [5]. If BBSA are to be used in the experiment, an aliquot of beads is removed at this time. 9. The BBSA are opsonized with antiBSA IgG. Prepare a stock solution of antibody (6 mg IgG/ml PBS). The volume of buffer and amount of antibody is proportional to the number of beads being opsonized. For example, 40 µg antibody stock solution is used to opsonize 50 mg beads (∼2 × 109 beads) in 1 ml PBS. Beads are mixed by inversion for 45 min and then washed two times with 1 ml PBS. 10. Following the final wash, the concentration of IgG-opsonized beads (BIgG) can be determined with a hemocytometer. Dilution is recommended to obtain an accurate bead count. 15.3.2 Protocol B: Preparation of IgGOpsonized Sheep Red Blood Cells (SRBC) Production of opsonized-fluorescent SRBC is also a three-step process:
15.3.2.1
Sheep red blood cells: SRBC (Cat. No. 212388; Becton Dickinson, Franklin Lakes, NY; http://www.bd.com/) Veronal buffered saline (VBS, Cat. No. 12-624E; Cambrex Bio Science Walkerville, Inc., http://www.cambrex.com/) Rabbit Anti-SRBC IgG: Rabbit IgG Fraction to Sheep Red Blood Cells (Cat. No. 55806; ICN Biochemicals, Inc.) R-phycoerythrin (PE) conjugated goat F(ab′)2 Anti-Rabbit IgG(H+L) antibody (Cat. No. L43004; CalTag Laboratories, Burlingame, CA; http://caltag.com/) 15.3.2.2 1.
2. 3. 4.
1. 2. 3.
Washing the SRBC Opsonizing the SRBC with rabbit anti-SRBC IgG Labeling the opsonized SRBC with tracer amounts of R-phycoerythrin (PE) conjugated goat anti-rabbit IgG
Red blood cells are fragile. Aspirate the supernatant from pelleted cells. Gently flick the bottom of the tube to dislodge pelleted cells and then add buffer. Vigorous vortexing will lyse the cells. 228
Materials and Reagents
5. 6.
Labeling Procedure
Wash the SRBC. Remove 1.5 ml SRBC from the Becton Dickinson stock solution into a 15-ml conical tube. Add 10 ml VBS. Mix by inversion, flicking the bottom of the tube if necessary to completely resuspend the cells. Pellet the cells by centrifugation at ∼700 × g for 5 min at 4°C. Aspirate the supernatant and replace with 10 ml VBS. Repeat Steps 2 and 3 until the supernatant is relatively clear (i.e., free of lysed SRBC as evidenced by the absence of a red tinge to the solution). When the supernatant appears relatively clear, remove the supernatant and replace with 7 ml VBS. Determine the concentration of blood cells spectrophotometrically. Using a water blank, measure the absorbance of a 1:25 dilution of the washed red cells at 541 nm (A541).
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8. 9.
10. 11. 12.
13. 14.
For example, for 2.5 ml, dilute 100 µl washed red cells with 2.4 ml H2O. The A541 of the diluted SRBC should be about 0.42 (0.38–0.46). This absorbance corresponds to a concentration of ~1 × 109 SRBC/ml. Adjust the volume of the solution if the A541 is not within these limits. To calculate the correct final volume, multiply the A541 by the volume of washed cells. Divide this number by 0.42 and add VBS to this calculated volume. After adjusting the volume, leave the washed SRBC at 4°C overnight. These “rested red cells” are less likely to lyse during the opsonization procedure than those opsonized immediately after washing. Dilute 1 ml SRBC with 948 µl VBS. While gently vortexing this suspension of SRBC, add 52 µl rabbit antiSRBC IgG and incubate the cells for 40 min at room temperature. Vortexing at this step is essential to generate uniformly opsonized targets. Wash three times with 1 ml VBS and resuspend the cells in 2 ml VBS. Remove 1 ml of the suspension and place into an amber 1.5-ml microcentrifuge tube. While gently vortexing, add 3.33 µl R-phycoerythrin (PE) conjugated goat F(ab′)2 anti-rabbit IgG(H+L). Note: Minimize light exposure from this point on, as this antibody is light sensitive. Incubate for 30 min on ice in a cold room in the dark. Wash three times with 1 ml VBS. After the final wash, remove the supernatant and resuspend the pellet in 1 ml VBS. The final concentration of cells is ~5 × 108/ml.
15.3.3 Protocol C: Transient Transfection of RAW Cells For imaging live RAW cells, transfections are done in glass-bottomed MatTek chambers. These chambers are constructed from standard size 35-mm disposable plastic Petri dishes that have a hole in the center of the dish which is sealed with optical-quality glass for high-resolution microscopic imaging using an inverted microscope stand. The small well thus created provides a convenient incubation chamber for the in situ transfection of cells that are grown on this glass surface. It is assumed that the plasmid to be transfected has been purified and that the concentration is known. Vectors for the construction of GFP-cDNAs are available from BD Biosciences Clontech (http:// www.bdbiosciences.com/clontech/). 15.3.3.1
Materials and Reagents
MatTek chambers (Cat. No. P35GC-014-C, MatTek Corp., Ashland, MA; http://www.glass-bottom-dishes.com/) The RAW lacR/FMLPR.2 subclone of the mouse macrophage cell line RAW 264.7 was obtained from Dr. Steven Greenberg [6]. Hereafter, this cell line is referred to as RAW. In our hands, these cells are more efficiently transfected than the parental line available from the ATCC (American Type Tissue Culture Collection, Manassas, VA; http://www.atcc.org) (15 to 30% transfection efficiency for the subclone vs. less than 5% for the parental line). Cells are maintained in RAW cell media (see below) and passed at 80% confluency using trypsin. RAW cell media: D-MEM (Cat. No. 10013-CV; MediaTech, Herndon, VA; http://www.cellgro.com/), 10% heatinactivated bovine calf serum (Cat. No. SH30072.03, Hyclone, Logan, 229
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Molecular Morphology in Human Tissues: Techniques and Applications UT; http://www.hyclone.com/), 1% Pen-Strep (Cat. No. 30-002-CI; MediaTech). Serum is heat inactivated by incubation at 56°C for 30 min. Superfect (Qiagen Inc., Valencia, CA; http://www1.qiagen.com/) HBSS- -: 50 ml of 10X Hank’s Balanced Salt Solution (Cat. No. 14185-052, GIBCO Carlsbad, CA; http://invitrogen.com/) to which is added 2.3 ml of 7.5% NaHCO3 and 5 ml of 1 M HEPES. The pH is adjusted to 7.4 with 10 mM NaOH (~2.5 drops) and diluted to 500 ml with dH2O. Filter sterilize. HBSS++: 500 ml HBSS - - containing 1.55 mM each of CaCl2 and MgCl2. Filter sterilize. 15.3.3.2 1.
2.
230
3.
4. 5.
6.
Transfection of Macrophages
Cells can be plated in the MatTek dish the day before transfection (2 × 105 cells/2 ml RAW cell media) or the same day (4 × 105 cells/2 ml RAW cell media), providing time for the cells to attach prior to transfection. Preparation of the transfection solution. For each DNA sample, 1 µg DNA is complexed with 2 µl Superfect in DMEM lacking antibiotics and serum (base media). Total volume/sample = 50 µl. Increase proportionately for multiple samples. Place DMEM in a 1.5-ml microfuge tube. Add the DNA first, followed by the Superfect. As Superfect can bind to the plastic tube, care should be taken to ensure that it is added directly to the media and does not touch the sides of the tube. Vortex the mixture and let the DNA and Superfect complex at room temp for 10 to 12 min. Note that incubation times greater than 30
7.
.
min significantly decrease the efficiency of transfection. While the DNA and Superfect are complexing, wash cells twice with 3 ml HBSS - - and leave the cells in the last wash until the DNA is complexed. After the DNA is complexed, add 150 µl DMEM base media to the tube and pipet to mix. Completely remove the buffer from the cells by aspiration. Add the complexed DNA to the glass well in the MatTek chamber; 200 µl fills the well. If a larger volume is added, it will spill out of the well and leave the center of the well dry. Carefully move the chambers to a 37°C incubator. For multiple samples, we have found it easier to transport them in 15-cm Petri dishes. Leave the DNA on the cells for 5 to 6 hr. Remove the media, replace with 2 ml RAW cell media, and leave overnight to allow plasmid to be expressed. Total time from addition of DNA to imaging is 16 to 24 hr. Imaging the day after transfection gives the highest signal. After 24 hr, the signal progressively decreases.
15.3.3.3 Imaging of RAW Cells that Are Expressing GFP-Protein Kinase Cs Phagocytosis of IgG-coated glass beads or opsonized SRBC can be followed over time using an inverted confocal laser scanning fluorescence microscope and a 40×, 1.3 NA oil objective lens. Any make of confocal microscope can be used; however, we used a Zeiss 410 confocal laser scanning microscope (LSM) (Carl Zeiss MicroImaging, Inc, Thornwood, NY; http://www.zeiss.com/). The GFP was visualized using 488-nm argon excitation and a 505–550 bandpass emission filter, and the Alexa 568 was detected using 543-nm HeNe excitation and a 560
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Visualization of Signal Transduction Pathways in Real Time longpass emission filter. The confocal “pinhole” should be adjusted to produce a relatively thick optical section so that the plasma membrane of the macrophage and the target do not move out of the optical section during active phagocytosis. The choice of “pinhole” diameter is a compromise between adequate signal detection and axial resolution. Experiments were performed at room temperature to slow the rate of phagocytosis. Consequently, the reported measurements of the rates of the uptake of fluorescent targets and of the translocation of the GFP-PKCs are normalized to room temperature by default. For purposes of illustration of the protocol, we used IgG-opsonized glass beads (BIgG) as the targets for phagocytosis. However, any fluorescent particle/pathogen can be used, providing its fluorescence does not overlap that of GFP. 1. 2.
3. 4.
Just before imaging, remove the media from the cells and add 800 µl of room-temperature HBSS++. Locate the cells to be imaged using the wide-field fluorescence mode. Set up a time-lapse program to collect an image every 10 sec over a period of 10 min (60 images total). Dilute 2 × 106 BIgG targets in 200 µl HBSS++. Switch to confocal mode and, while imaging the cells expressing GFPconjugated PKCs in real time, add beads (4:1, BIgG:macrophage) directly over the cells and start the timelapse program as soon as the beads are in focus.
15.4 RESULTS OF THE VISUALIZATION OF TRANSFECTION OF RAW CELLS The power of real-time imaging lies in the wealth of data that is contained within the time-lapse movies. Initially, it can be
used to identify proteins that accumulate during a localized event. For phagocytosis, the concentration of GFP-conjugated signaling molecules around the targets implicates them either in target uptake or in subsequent signaling events. For example, PKC-ε, but not PKC-δ, translocates and concentrates at forming phagosomes, and was identified as a component of the uptake machinery (Figure 15.1 and Larsen et al. [2]). Once a signaling protein has been identified as participating in a process, truncation or deletion mutants can be tested to determine which protein domains are involved in translocation. Although real-time imaging provides animated views of protein translocation, this information can also be obtained with fixed time point assays, in which phagocytosis is synchronized and cells are fixed at intervals between 0 and 15 min [5]. However, real-time imaging allows one to follow the phagocytosis of individual particles over time, to quantify not only the rate of ingestion (time/bead), but also the localization time (i.e., the time the GFP constructs are associated with targets). 15.4.1 Rate of Ingestion The rate of ingestion is measured from the first indentation of the plasma membrane until the target is encircled. Times are determined by subtracting the time at which the first indentation of the membrane is observed from the time at which the particle is completely encircled with GFP. Examination of the fluorescence images in the green channel alone (GFP fluorescence) makes determination of the time of first indentation and of complete engulfment easier (Figure 15.1, lower row, panel 3). For Figure 15.1, internalization of the particle in PKC-ε expressing cells was 128.284 − 78.856 = 49.43 sec and 312.016 − 240.196 = 71.82 sec for PKC-δ. 231
Figure 15.1 (Color Figure 15.1 follows page 106.) Quantitation of the rate of phagocytosis and of PKC accumulation using real-time confocal microscopy. RAW cells were transfected with 1 mg GFP PKC-ε (upper) or GFP PKC-δ (lower). Images were taken at 10-sec intervals for 10 min following addition of Alexa 568-labeled BIgG. Frames were chosen to illustrate ingestion and accumulation of the indicated particle. (e) PKC-ε panel: 1 – binding, 2 – first accumulation, 3 – ingestion complete, 4 – loss of concentration. (Arrow in panel 2 indicates time of first accumulation of red target.) (d) PKC-δ panel: 1 – binding, 2 – ingestion complete. Time for ingestion: PKC-ε: 49.43 sec, PKC-δ: 71.82 sec. PKC-ε accumulation: 137.71 sec. Evaluating the green signal alone facilitates determination of time of complete ingestion. PKC-δ: panel 3, is the first frame in which the bead (indicated by the arrow) is completely surrounded by green. Note the absence of accumulation of GFP around the engulfed target. This is clearly seen in PKC-δ panel 4, where the application of a linear lookup table (LUT) demonstrates that PKC-δ does not accumulate at targets during phagocytosis. The linear LUT is an extended topographic color map that is divided into 24 colors. This pseudocolor LUT visually highlights the relative concentration of the GFP-PKC-δ. Cool color shades (e.g., blue, green) represent low concentrations, while shades of hot colors (e.g., red, white) represent higher concentrations; blue being the lowest and white the highest concentration. Videos of the both experiments are available at http://www.jcb.org/cgi/content/full/jcb.200205140/DC1.
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Visualization of Signal Transduction Pathways in Real Time Thus, expression of PKC-ε decreases the amount of time required for internalization (i.e., increases the rate of uptake). Quantifying the rate of ingestion from several experiments confirmed this data. Uptake of IgG-opsonized particles in control (GFP transfected) cells was 80 ± 5 sec/bead (n = 69), in cells expressing GFP PKC-ε was 35 ± 2 sec (n = 35), and in GFP PKC-δ expressing cells was 76 ± 4 sec (n = 49) [2]. 15.4.2 Localization Time Localization time is calculated from the initial concentration of GFP at the site of uptake (as determined by pixel density, see below) until the pixel density returns to background levels. For the cell shown in Figure 15.1ε, the localization time is 137.71 sec (226.425 − 88.713). This measurement can be used to study the stability of the membrane/PKC-ε interaction and identify factors (i.e., drugs, mutation of putative membrane binding regions, or upstream or downstream modulators) that alter that association. The technique can be applied to any fluorescent molecule that concentrates on a membrane or on a specific organelle.
for variations in GFP expression levels and for any baseline association of the expressed protein with the membrane. Also, the confocal nature dictates that each image is the same thickness. Thus, an increase in signal results from the accumulation of GFP-protein rather than “edge effects” that might be detected if more membrane is recruited to the targets. The Iphagosome/Imembrane ratio of a GFP-protein that does not accumulate will be ~1, in contrast with wild-type PKC-ε, where the ratio is 2 to 3. For the cell shown in Figure 15.2, bead A has a normalized intensity of 2.3 and the intensity of bead C is 1.4. Variation in pixel density can also be effectively presented visually using a linear LUT (lookup table) (Figure 15.2d). Some mutants may localize weakly with the phagosomes and their accumulation will be lower than that seen with wild-type PKC-ε. Thus, the inherent high sensitivity of the confocal microscope is critical to the success of these experiments. The sensitivity of the system is illustrated in Figure 15.2. Although particle C is 30% as bright as particle A, both are significantly above the fluorescence of the uninvolved membrane. Thus, a 70% decrease in intensity can be easily detected with this method.
15.4.3 Quantitation of GFP Accumulation PKC localization, measured as GFP accumulation, is quantified by creating a pixel intensity profile through the phagosome to a region of plasma membrane devoid of bound targets (Figure 15.2c). Pixel density is calculated on a relative scale (Figure 15.2). The fluorescence intensity at the phagosome (Figure 15.2a: A, C) is normalized to the fluorescence intensity at a portion of the plasma membrane that is not involved in uptake of a target (Figure 15.2a: B) and reported as the ratio (Iphagosome/Imembrane). This strategy compensates
15.5 PROTOCOLS FOR THE VIRAL TRANSDUCTION OF HUMAN AND MOUSE MACROPHAGES Because macrophages are difficult to transfect, molecular studies in this cell type have lagged behind those of more readily transfectable cell types such as fibroblasts. The mouse macrophage cell line RAW 264.7 has been the most amenable to molecular manipulation and is the cell type of choice for studies on phagocytosis and gene regulation (for example, see [7–11]). 233
Figure 15.2 (Color Figure 15.2 follows page 106.) Quantitation of accumulation of GFP at targets. The cell is transfected with GFP-PKC-ε. (a) and (b) show the GFP (green) channel only; however, (b) is pseudocolored with a linear LUT (see legend for Figure 15.1) and visually shows the differential concentration of GFP around beads at arrows A and C, and the absence of concentration at point B, an uninvolved region of the plasma membrane. (d) is a merged image of the same cell showing Alexa 568 and GFP staining. (c) is a plot profile of the pixel intensity for GFP along the line drawn on the cell in (a) determined using the NIH ImageJ program. Accumulation was normalized to position B and calculated as 2.3 for point A, and 1.4 for point C. That these points correspond to the highest concentration for each bead is confirmed in the pseudocolored image. The GFP intensity is not zero at point B because there is GFP-PKC-ε in the cytosol adjacent to the membrane; however, there is no significant accumulation at this region of the plasma membrane.
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Visualization of Signal Transduction Pathways in Real Time Human monocytic cell lines have been even more refractory to transfection [1]. The low transfection efficiency of these cells makes population-based studies (such as regulation of respiratory burst and secretion of cytokines) very cumbersome, requiring that the transfected cells be collected by cell-sorting to obtain a population in which the majority of the cells are expressing the gene of interest. To overcome such problems, viruses have been used to deliver DNA to hard-to-transfect cells. Macrophages, however, also appear to be relatively refractory to viral transduction. We tried unsuccessfully to introduce DNA into Mono Mac-6 cells, the most differentiated of the human monocytic cell lines [12] using adenovirus and Sindbis virus. However, ViraPower Lentiviral Expression from Invitrogen has provided a system for generating stable Mono Mac-6 cell lines that express GFP-conjugated proteins. This has permitted us to expand our studies on the involvement of PKCs in cell signaling to human macrophages. Below we present protocols for the construction of the viral plasmid, for the generation of virus, and for the transduction and characterization of the resulting cell lines.
http://www.cellgro.com/), 10% heatinactivated fetal bovine serum (Cat. No. SH30070.03; Hyclone, Logan, UT; http://www.hyclone.com/), 2 mM L-glutamine (Cat. No. 25-005CI; MediaTech), 1% Pen-Strep (Cat. No. 30-002-CI; MediaTech), 500 µg/ml Geneticin (Cat. No. 11811023; Gibco, Carlsbad, CA; http:// invitrogen.com/) T-150 tissue culture flask (Cat. No. 430825, Corning) TOPO Expression kit: pLenti6/V5 Directional TOPO Cloning Kit (Cat. No. K4955-10, Invitrogen, Carlsbad, CA; http://invitrogen.com/) ViraPower Lentiviral Expression System (K4950-00, Invitrogen, Carlsbad, CA) ViraPower Lentiviral Support Kit (Cat. No. K4970-00, Invitrogen, Carlsbad, CA) Corning syringe filter: 0.45-µm filter (Cat. No. 430571, Corning Life Sciences; http://www.corning.com/lifesciences/) 15.5.1.2 Construction of Viral Plasmids 1.
15.5.1 Protocol D: Viral Plasmid Construction Here we describe construction of the lentiviral plasmids. Although the basic protocol will be similar for insertion of any cDNA, the specifics (i.e., restriction sites used for insertion and diagnostic digests) will vary with the insert. 15.5.1.1
Materials and Reagents
293FT media: D-MEM (Cat. No. 10013-CV; MediaTech, Herndon, VA;
2.
3. 4.
To provide a site for the insertion of the cDNA of interest, remove the lacZ insert in the pLenti6/V5GW/lacZ plasmid (in TOPO Kit) by digestion with Asc I and Spe I. We subcloned our cDNAs (GFP, GFP conjugated PKC-ε, and GFP conjugated PKC-δ) into the vector using the Asc I and Spe I site. Perform a diagnostic RE (restriction enzyme) digest to ensure that the resulting plasmids contain insert. Digestion with AflII was done to ensure that the LTRs of the vector were intact. LTRs are necessary for integration of the DNA into the host 235
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5.
cell genome. As there is one AflII site in each LTR, two bands should be generated if the LTRs are intact and the insert does not have an AflII site. At this point, it is recommended that you confirm the sequence of the insert. Alternatively, the plasmids can be expressed in mammalian cells (we used 293-fibroblasts) and expression of the protein confirmed by Western blot analysis.
15.5.1.3 Stock
Production of a Lentiviral
The virus is produced by co-transfecting the cDNA-containing pLenti plasmid and a packaging plasmid mix (Lentiviral Support Kit) into the 293FT packaging cell line (TOPO Expression Kit). Productively transfected cells produce virus that will be released into the media. The media is harvested and the virus concentrated by centrifugation. The day prior to transfection, trypsinize and count 293FT cells (supplied in the Invitrogen Lentiviral Expression kit). Plate 1 × 107 cells per T-150 flask in 40 ml of complete 293FT media. Two control flasks are needed for each experiment: one that will not be transfected (negative control) and one that will transfected with the pLenti6/V5-Gw/lacZ plasmid (positive control) supplied with the TOPO kit. 2. On the day of transfection, remove the media and replace it with 22 ml of 293FT media lacking antibiotics plus 10 ml of 293FT media lacking antibiotics and serum. 3. Transfection: - Combine 18 µg optimized packaging mix (in Lentiviral Support kit) and 6 µg pLenti6/V5-GW express-
4.
1.
236
5.
6. 7. 8. 9.
ing the cDNA of interest in 1.5 ml of 293FT media lacking serum and antibiotics. Mix gently. - Dilute 72 µl Liptofectamine 2000 (in Lentiviral Support kit) in 1.5 ml of 293FT media lacking serum and antibiotics. Mix gently. - Incubate at room temperature for 5 min. - After incubation, combine the DNA/packing mixture (Step 3a) with the Liptofectamine 2000 solution (Step 3b). Mix gently and allow them to complex at room temperature for 20 min, shaking gently every 5 min. - Carefully add the DNALiptofectamine complex to the T-150 flask of cells. Note that the cells are very fragile and will not transfect if they come off the plate. Mix by gently rocking flask back and forth. Leave flasks in incubator for 24 hr. After 24 hr, remove the media and replace it with 35 ml of 293FT media without Geneticin. By 24 hr, the cDNA is being expressed. In our case, the protein was GFP conjugated so transfection could be detected by fluorescence microscopy. Collect the cell media 48 hr posttransfection (24 hr post media change). Replace with 35 ml of 293FT media without Geneticin. Cells may lift off the plate in some areas. Work carefully to retain as many adherent cells as possible. Return cells to incubator for 24 hr at 37°C. Centrifuge the harvested media (400 × g for 5 min at 4°C) to remove cells. Filter the media through a 0.45-µm low protein binding Corning filter. Freeze this virus-containing media at −20°C overnight. Sterilize the buckets and tubes for a Beckman SW-28 rotor. To do this,
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Visualization of Signal Transduction Pathways in Real Time rinse tubes and buckets with 70% EtOH and then rinse twice with sterile PBS. Dry in a tissue culture hood overnight without the UV light, as the UV light will damage the tubes. 10. Harvest a second batch of virus 72 hr post transfection (24 hr post media change). Repeat Step 7; then proceed to concentration of the virus. 15.5.1.4 of Virus
Concentration and Titration
This is a modification of a published protocol [13]. 1.
2. 3. 4. 5. 6.
7.
Thaw the virus-containing media on ice, combine it with the second batch harvested in Step 10 above, and transfer it to sterile Beckman tubes (35 ml per tube). Put the tubes into the sterile buckets. Pellet the virus by centrifugation (Beckman SW- 28, 100,000 × g, 1.5 hr, 4°C). Immediately remove the media and add 500 µl of 293FT media lacking Geneticin. Carefully resuspend the virus with a pipette, avoiding bubbles to prevent clumping. Incubate at 4°C for 1 hr. Aliquot virus stocks and store at −80°C. Store in small aliquots, as repeat freeze–thawing will decreases the viral titer. Titer the virus exactly as detailed in ViraPower the Invitrogen Lentiviral Expression System (Version B) Instruction Manual. In our hands, viral titers of ~5 × 106 TU (Titer Units)/ml were obtained after one thaw. Freshly concentrated virus gave a titer of 7–8.5 × 106 TU/ml.
15.5.2 Protocol E: Transduction of Human and Mouse Cells Using the following protocols, we were able to produce virus containing cDNAs for GFP, GFP-conjugated PKC-ε, and GFP-conjugated PKC-δ (Figure 15.3). Mouse RAW cells and human Mono Mac6 cells were used for our studies. 15.5.2.1
Materials and Reagents
The human monocytic cell line, Mono Mac-6, was supplied by the DSM German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; http://www.dsmz.de/). Cells are maintained in the complete MonoMac-6 media listed below. These cells
Figure 15.3 Expression of viral plasmids in human monocytic cells. Mono Mac-6 cells were transduced with lentivirus carrying cDNA for the indicated proteins (MOI = 100). Expression of GFP was seen 7 days post transduction. Transduced cells were selected with 2.5 mg/ml Blasticidin on day 7. Flow cytometry data reveals >80% GFP positive cells following selection for 7 days (i.e., 14 days post transduction). Arrows in each panel indicate the non-transduced cell population.
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Molecular Morphology in Human Tissues: Techniques and Applications do not grow well in sparse culture and have a doubling time of ~36 hr. They are normally seeded at 5 × 104/ml and will reach 1 × 106/ml in 7 days. Cells should be split 1:20 when the density reaches 7.5 × 105 to 1 × 106/ml. The media should be changed at least once a week, regardless of the cell density. Mono Mac-6 media: RPMI 1640 (Cat. No. 10-040-CV; MediaTech), 10% heat-inactivated bovine calf serum (Cat. No. SH30072.03; Hyclone), 2 mM L-glutamine (Cat. No. 25-005CI; MediaTech), 1% Pen-Strep (Cat. No. 30-002-CI; MediaTech), 1 mM sodium pyruvate (Cat. No. 25000CI; MediaTech), 0.1 mM nonessential amino acids (Cat. No. 25025CI; MediaTech), 9 µg/ml bovine insulin (Cat. No. 13007-018; Gibco) Costar 48-well TC-treated microplate (Cat. No. 3548, Corning Lifesciences) Blasticidin (Cat. No. R210-01, Invitrogen, Carlsbad, CA) 15.5.2.2 1. 2.
3.
238
4.
5. 6.
7.
Transducing Cells
Plate 1.2 × 104 cells/well in a 48-well plate in appropriate complete media. Let cells adhere at least 2 hr. Although Mono Mac-6 cells are usually cultured in suspension, they do adhere to the Costar brand 48-well plates. Remove the media from adherent cells and replace with media containing virus at an MOI (multiplicity of infection, the ratio of virus:cell) of 50 for the RAW cells and 100 for the Mono Mac-6 cells. A total of at least 300 µl should be placed in each well. If you have a low titer and your experiment requires more than 300 µl virus per well, the cells can survive in this media overnight.
8.
9.
At 24 hr post transduction, triturate the media to break up any cell clumps, and put this media into new wells. Add 1 ml complete media to the original wells and 700 µl media to the cells containing the spent media. Continue transduction for an additional 6 days, changing the media if necessary. On day 7, begin the selection process by replacing the media with media containing 2.5 µg/ml Blasticidin. From this point on, cells are maintained in this Blasticidin Selection Media. Expression of the transduced protein usually occurs on day 7 or 8. In our hands, expression was not detected before day 7. Figure 15.3 illustrates the expression of our three constructs following 7 days of Blasticidin selection. Split the cells when they reach 70 to 80% confluency by trypsinzation (50 µl/well, 5 min). Trypsin is quenched with 550 µl media containing Blasticidin. Expand the number of cells by plating 100 µl cell suspension in each of six wells of a 24-well plate. Add Blasticidin containing media to a final volume of 1 ml. Note: Mono Mac-6 cells will now be in suspension. Trypsinize RAW cells at 70 to 80% confluency. Combine like wells and expand into a T-25 flask. Mono Mac6 cells should be harvested 7 to 10 days after expansion. The exact time is empirical; cells should be relatively dense in the wells. Combine like wells, remove the spent media by centrifugation, and expand into T-25 flasks (10 ml). Positive cells are monitored by flow cytometry (FACScan; Becton Dickinson Immunocytometry System). Cells are evaluated twice a week and
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Visualization of Signal Transduction Pathways in Real Time the percentage of GFP-positive cells is recorded. 10. For flow cytometry, non-transduced cells were used to set the threshold for positivity. The gate was set such that 5% of the cells appeared positive for GFP. 11. It is recommended that aliquots of cells be frozen when the % positivity is greater than 80%.
15.5.3.1
Falcon 12 × 75-mm tubes (Cat. No. Krackeler Scientific, 352008, www.krackeler.com) LPS: E. coli lipopolysaccharide (Cat. No. L2630, Sigma-Aldrich) Brefeldin A (Cat. No. 194802, ICN Biomedicals, Inc.) Paraformaldehyde (Cat. No. P-6148, Sigma-Aldrich) Bovine serum albumin (BSA, Cat. No. BP1600-100; Fisher Scientific; http://fishersci.com/) Triton X-100: TX-100 (Cat. No. 28314; Pierce Biotechnology, Rockford, IL) R-Phycoerythrin conjugated anti-mouse TNF-α (Cat. No. RM9014; CalTag, Burlingame, CA)
15.5.3 Protocol F: Characterization of Transduced Cells by Dual Parameter Flow Cytometry Although lentivirus can be used to efficiently express cDNAs, it is necessary to demonstrate that the transduction per se does not alter the basic phenotype of the cells to be a viable tool for signal transduction research. To assess this, cell-specific marker functions in the transduced cells should be compared to the same functions in non-transduced cells. We specifically assessed two markers for phagocyte function: (1) phagocytosis and (2) expression of the cytokine, TNF-α, by Mono Mac-6 and RAW cells that were transduced to expressed GFP. We compared the level of phagocytosis and TNF-α production in non-transduced cells and cell populations in which more than 80% of the cells were positive for GFP. Dual parameter flow cytometry can be used to compare phagocytosis or TNF-α production on a cell-bycell basis. This approach allows one to determine if the level of expression of the transduced protein affects the function of interest and simultaneously facilitates the rapid collection of information on thousands of cells.
Materials and Reagents
15.5.3.2 1. 2. 3. 4. 5. 6. 7. 8.
IgG-Mediated Phagocytosis
Perform analysis in Falcon 12 × 75mm tubes containing 4 × 105 cells. Wash cells once in 500 µl HBSS++ and resuspend in 200 µl HBSS++. Transfer the tube to ice and add PESRBC targets at a ratio of 20 PESRBC/macrophage. Centrifuge gently (120 × g, 2 min, 4°C) to increase contact between macrophages and targets. Incubate tubes in a 37°C water bath for 1 hr to allow phagocytosis to occur. Add 1 ml of 0.83% ammonium chloride to lyse the uningested SRBC and mix well. Centrifuge the suspension (200 × g, 5 min, 4°C) and discard the supernatant. This removes the lysed SRBC. Resuspend the cell pellet in 500 µl PBS for analysis by flow cytometry. Set control green and red gates on
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Molecular Morphology in Human Tissues: Techniques and Applications non-transduced macrophages that were not presented with targets, such that 98% of the cells are contained in the lower left quadrant of the dot plot (GFP - green, PE - red ). 15.5.3.3 Results of Experiments on IgG-Mediated Phagocytosis Virally transduced GFP does not alter IgG-mediated phagocytosis in either Mono Mac-6 or RAW macrophages as determined by the fact that the extent of phagocytosis (i.e., the mean fluorescence intensity, MFI) is not altered upon expression of GFP: high GPF expressers have the same level of phagocytosis as the low and non-expressers (Figure 15.4). This is consistent with the
conclusion that viral transduction per se does not alter the ability of macrophages to undergo phagocytosis. 15.5.4 Protocol for TNF-α Production 15.5.4.1
Procedure
We present a protocol to assess LPSstimulated production of TNF-α by virally transduced GFP in RAW cells. Based on the results of the phagocytosis data, we anticipated that viral transduction would not affect LPS-stimulated production of TNF-α in Mono Mac-6 cells. The protocol presented should be applicable to other macrophages.
Figure 15.4 Viral transduction of GFP does not alter IgG-mediated phagocytosis in macrophages. The non-transduced RAW and Mono Mac-6 cells efficiently took up the PE-SRBC, as indicated by the degree of fluorescence intensity in the upper-left panels of the dot plots. No significant signal was detected in the green channel, as indicated in the upperright-hand panel of the dot plots. In contrast, for the GFP transduced cells, there was a significant fluorescence signal in both the red and green channels, as indicated in the upper-right panels of the dot plots. The similar MFIs of transduced and controls demonstrate that transduction per se does not alter the phagocytic capacity of the cells.
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Visualization of Signal Transduction Pathways in Real Time 1. 2.
3.
4. 5.
6.
Plate RAW cells the day prior to the experiment (5 × 105 cells/well in a 24-well plate). Stimulate cells for TNF-α production by the addition of LPS (100 ng/ml). To prevent TNF-α release from the cells, 10 µg/ml of Brefeldin A is added with the LPS. After 4 hr, replace the media with 1 ml PBS. Transfer the cells to ice and leave them on ice at 4°C for an additional 45 min to facilitate the removal of the cells from the plates. Scrape cells into Falcon tubes and centrifuge (200 × g, 5 min). Fix cells with 0.5 ml of 3.7% paraformaldehyde for 10 min at room temperature. Remove the formaldehyde and permeabilize the cells with 500 µl of 2% BSA in PBS containing 1% TX-100 for 10 min on ice in the dark. After 10 min, add 0.6 µl R-phycoerythrin conjugated anti-Mouse TNF-α
7.
and incubate on ice in the dark, in the cold, for 30 min. Limit exposure to light because this antibody is light sensitive. Centrifuge the cells (200 × g, 5 min) to remove unconjugated antibody and resuspend in 500 µl PBS for analysis by flow cytometry. Gate the samples on RAW cells that received Brefeldin A but no LPS or antibody, such that 98% of the cells are contained in the lower left quadrant (GFP - green, PE - red).
15.5.4.2
Results of TNF-α Production
Virally transduced GFP does not alter LPS stimulated TNF-α production in RAW macrophages as determined the MFI for non-transduced and GFP transduced cells are essentially equivalent (108 vs. 112, Figure 15.5). As with phagocytosis, high GPF expressers have the same level of TNF-α production as the low expressers.
Figure 15.5 Viral transduction does not alter the ability of RAW cells to synthesize TNF-α in response to LPS. Non-transduced control RAW cells (left) and those stably expressing GFP (right) were stimulated with 100 ng/ml LPS. To prevent release of TNF-α, Brefeldin A (10 mg/ml) was added with the LPS. At 4 hr, the cells were fixed, permeabilized, and stained for intracellular TNF-α using an anti-mouse TNF-α antibody directly conjugated to PE. Both transduced and control cells produced TNF-α, as demonstrated by fluorescence in the upper quandrants. The MFI in the upper-left quadrant of the control cells is equivalent to that in the upper right (GFP expressing cells) of the transduced cells, demonstrating that viral transduction per se does not alter the ability of RAW cells to produce TNF-α.
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Molecular Morphology in Human Tissues: Techniques and Applications These results are also consistent with the conclusion that viral transduction per se does not affect the ability of macrophages to synthesize cytokines in response to LPS. 15.6 DISCUSSION The use of molecular biology to probe the cell biology of macrophages has lagged behind that of many other cell types because of the difficulties in transfecting hematopoietic cells. Recent advances in lipid transfection and the availability of kits for construction of viruses for transduction have provided tools for studying unique macrophage functions. This chapter has detailed the procedures for transient transfection and viral transduction of GFP-conjugated signaling molecules into mouse and human macrophages. From real-time confocal time-lapse movies, we have shown how one can calculate (1) the duration of GFP conjugated protein concentrate, (2) where it concentrates, and (3) the increase in concentration compared to non-involved regions of the membrane. Lentiviral transduction affords researchers the ability to generate human macrophage cell lines that stably express proteins of interest. We have used dual parameter flow cytometry to demonstrate that viral transduction per se does not affect the ability of macrophages to take up antibody-opsonized particles nor to synthesize cytokines in response to bacterial lipopolysaccharide. These techniques can be applied to a variety of cell types and processes that will add to our understanding of basic cell function.
50821 (MRL). MRL received a visiting professorship from the Biosignal Research Institute of Kobe University in Kobe, Japan, to perform the confocal experiments reported herein. We wish to express our gratitude to Dr. Naoaki Saito and the members of his research group in Japan for providing their expertise, protocols, and use of the Zeiss 410 LSM. We are also indebted to Peter Welch and Kenneth Frimpong of Invitrogen’s Research and Development team. Their help in troubleshooting and providing reagents was invaluable to the success of the Lenti virus project. Finally, we wish to thank Drs. Timothy Sellati and Daniel Loegering for their help in setting up the TNF-α assays. Quantitative analyses of fluorescence intensity (Figure 15.2) were performed on a Windows XP computer using the public domain ImageJ program developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij/.
References 1.
2.
3.
4.
5.
Acknowledgments
The work presented here was supported in part by a research grant from the National Institutes of Health RO1 GM242
6.
Sanchez-Mejorada, G. and Rosales, C., Fcgamma receptor-mediated mitogen-activated protein kinase activation in monocytes is independent of Ras, J. Biol. Chem., 273(42), 27610, 1998. Larsen, E.C. et al., A role for PKC-ε in FcγRmediated phagocytosis by RAW 264.7 cells, J. Cell Biol., 159, 939, 2002. Garcia-Garcia, E. and Rosales, C., Signal transduction during Fc receptor-mediated phagocytosis, J. Leukoc. Biol., 72, 1092, 2002. Greenberg, S. and Grinstein, S., Phagocytosis and innate immunity, Curr. Opin. Immunol., 14, 136, 2002. Larsen, E.C. et al., Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells, J. Immunol., 165, 2809, 2000. Cox, D. et al., Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes, J. Exp. Med., 186, 1287, 1997.
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Visualization of Signal Transduction Pathways in Real Time 7.
Diaz-Guerra, M.J.M. et al., Up-regulation of protein kinase C-ε promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells, J. Biol. Chem., 271, 32028, 1996. 8. Bajno, L. et al., Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation [see comments], J. Cell Biol., 149, 697, 2000. 9. Marshall, J.G., Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis, J. Cell Biol., 153, 1369, 2001. 10. Lee, D.J. et al., Rac1 and Cdc42 are required for phagocytosis, but not NF-kappa-B- dependent gene expression, in macrophages challenged with Pseudomonas aeruginosa, J. Biol. Chem., 275, 141, 2000.
11. Zhang, Q. et al., A requirement for ARF6 in fcγ receptor-mediated phagocytosis in macrophages, J. Biol. Chem., 273, 19977, 1998. 12. Ziegler-Heitbrock, H.W.L. et al., Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes, Int. J. Cancer, 41, 456, 1988. 13. Bartz, S.R., Rogel, M.E., and Emerman, M., Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control, J. Virol., 70, 2324, 1996.
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Proteolytic Activity Demonstrated by Film In Situ Zymography (FIZ): A Clinically Applicable Double-Staining Method Also Involving ImmunoGoldSilver Staining Masahiko Zuka
16.1 INTRODUCTION Expression of matrix metalloproteinases (MMPs) provides a reliable indicator of ongoing tissue remodeling. In particular, gelatinase A (MMP-2) and B (MMP-9) have attracted considerable attention in normal tissue events and also in various pathological conditions, including inflammation, metastasis, and angiogenesis. A new double-staining method, termed in situ zymography (ISZ), may lead to new directions in individualized medicine. The new technique was originally introduced to detect the activity of other kinds of enzymes, such as plasminogen activator, at that time mainly using animal tissue. It is principally based on the visualization of digested substrate on/beneath the fresh tissue section [1]. Pathological conditions in which MMP2 and MMP-9 are of particular interest include inflammation, metastasis, and angiogenesis. Clinical applications have not yet been introduced. MMPs are pro0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
duced and secreted as latent-form “zymogens” that require endopeptic cleavage of a propeptide domain to be activated. There are some inhibitors well known as tissue inhibitors of metalloproteinases (TIMPs) specific to MMPs. Not only in the proforms of the gelatinases, but also in some activated forms of them, MMP could be prevented from degrading extracellular matrix by these inhibitors. It is still difficult to find antibodies capable of discriminating between the active and inactive forms of these enzymes. Hence, for functional analyses of MMPs, combining immunohistochemistry with quantitative analysis of mRNA and protein would be required. As far as MMPs are concerned, the estimation of the activity of the enzyme is of primary interest. Therefore, MMPs provide a good example to illustrate the limitation of immunohistochemistry when used to identify a specific location and at the same time quantify specific MMP activities in tissue sections [2].
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Molecular Morphology in Human Tissues: Techniques and Applications The activities of two molecular species of gelatinases in human tissues have been analyzed using ISZ with a substrate containing a radioisotope [3, 4]. Recently, a number of articles on nonradioactive methods for detection of activity of MMPs have been published [5, 15]. However, these procedures are associated with some difficulties. During the incubation process, the substrate tends to fall off the glass slide, and it is problematic to localize cells and enzyme activity by comparing consecutive sections. Moreover, human tissue materials obtained by biopsy can sometimes be too scanty to allow estimation of proteolysis in clinical use (loss of these substances inhibits analysis on serial tissue sections). Film in situ zymography (FIZ) offers improvements achieved by the highbridging structure of gelatin substrate, supported by polyester film, in effect leading to a more successful and tighter fixation on the glass slide [16–22]. This tight adhesiveness makes it possible not only to check the location of the gelatinolysis activity, but also to perform double staining involving immunohistochemistry (IHC) [23]. Most of the problems stated above can be resolved using this new method. This chapter provides an introduction on how to perform the double staining of FIZ and IHC. This method yields a red color using Ponceau 3R for gelatin substrate, and a black color achieved by application of immunogold-silver staining (IGSS) with its high sensitivity and contrast against unstained background tissue [6, 25, 26]. Potential clinical applications are also discussed.
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16.2 PROTOCOLS FOR FILM IN SITU ZYMOGRAPHY DOUBLE STAINING 16.2.1 Protocol A: Incubation of FIZ Slides 16.2.1.1 Materials and Reagents Fresh human tissue embedded and frozen in OTC compound (TissueTek, Cat. No. 4583, Sakura Finetech, Torrance, CA) FIZ gelatin film: cross-linked gelatin films with a thickness of 7 µm supported by polyester film (“MMP in situ ZymoFilm,” Cat. No. 295-58001, WAKO Chemical, Osaka, Japan) Moist chamber 16.2.1.2 1.
2.
3.
Incubation Procedure
Fresh human tissues should be embedded in OTC compound, immediately frozen on dry ice methanol, and stored at −80°C. Prepare serial frozen sections with a thickness of 6 µm on a cryostat (e.g., Miles, Elkhart, IN) and mount them onto FIZ gelatin film as described above. Incubate the sections in a moist chamber at 37°C for the proper duration (also see Discussion).
16.2.2 Protocol B: Immunogold-Silver Staining 16.2.2.1 Materials and Reagents Immunogold, goat anti-mouse IgG antibodies adsorbed to 1-nm colloidal gold particles (AuroProbe One,
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Proteolytic Activity Demonstrated by Film In Situ Zymography (FIZ) GAM, Cat. No. 471, Amersham Biosciences, Buckinghamshire , U.K.) Bovine serum albumin (BSA fraction V, Cat. No. 05488, Sigma, St. Louis, MO) Buffer 1 (washing buffer): 0.8% bovine serum albumin (BSA) in PBS (Buffer 3) Buffer 2 (blocking solution): 0.8% BSA, 0.1% IGSS quality gelatin (Cat. No. 416; Amersham Biosciences), 5% normal goat serum (Cat. No. 426041, Nichirei, Tokyo, Japan), and 2 mM NaN3 in PBS Buffer 3 (PBS): phosphate buffered saline: 10 mM Na2PO4/NaH2PO4, 150 mM NaCl, pH 7.4 Primary antibody: anti-human alpha smooth muscle actin antibodies (1A4, Cat. No. 0851; DakoCytomation, Glostrup, Denmark) Silver Enhancement: Silver acetate autometallography according to Hacker et al. can be applied [25], as well as “IntenSE M” (Cat. No. 491, Amersham Biosciences), or the new “GoldEnhance” developer (Nanoprobes, Inc., Yaphank, NY) — the latter one leading to a very distinct and smooth staining Post-fixative: 2% glutardialdehyde (e.g., from TAAB Laboratories Equipment, Berkshire , U.K.) 16.2.2.2 1. 2. 3. 4. 5.
Staining Procedure
Wash in washing buffer (buffer 1) for 10 min. Incubate with blocking solution (buffer 2) for 30 min. Incubate with primary antibody for 90 min at room temperature. Wash in washing buffer, 3 times 10 min each. Incubate with immunogold reagent, diluted 1:50 in PBS containing 1% BSA.
6.
Wash in washing buffer, 3 times 15 min each. 7. Wash in PBS, 3 times 5 min each. 8. Wash in distilled water, 3 times 5 min each. 9. Postfix in glutardialdehyde solution for 10 min. 10. Silver enhance (this usually takes 5 to 15 min, depending on the developer protocol applied). Evaluate the protease activity with a light microscope (LM). Under optimal conditions, the reaction product seen in the LM first appears as small black dots, and finally a strong black color without nonspecific background staining should be reached. 11. Wash in distilled water 3 times, 5 min each. Optionally, you can select a prefixation using 4% buffered formaldehyde followed by ethanol (70, 90, 95, and 100%), ethanol/ xylene (1:1), pure xylene, and then ethanol (100, 95, 90, and 70%), distilled water to reach at washing buffer backwardly (recommendation of Prof. G.W. Hacker, in Salzburg). Caution: It is not recommended to use any nuclear counterstain because this could inhibit the quality and reading of the following secondary stain, which is expected to be fully clear. 16.2.3 Protocol C: Staining FIZ Slide with Ponceau 3R 16.2.3.1 Materials and Reagents 0.8% Ponceau solution (Ponceau 3R, Cat. No. 217522; ICN Biomedicals, Aurora, OH) in 6% trichloroacetic acid (Cat. No. T-6399; Sigma, St. Louis, MO)
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Molecular Morphology in Human Tissues: Techniques and Applications 16.2.3.2 1. 2. 3. 4. 5. 6.
Staining Procedure
70% ethanol, 30 min Ponceau 3R solution, 20 min 70% ethanol, 30 min 95% ethanol, 10 min 100% ethanol, 60 min Xylene, 30 min or longer
Option: You can add one additional step of Ponceau/ethanol solution (3:7 mixture) for 1 min between the Steps 1 and 2. 16.3 RESULTS The detection protocols proposed here were tested with an epitope of well-known distribution (Figure 16.1). Atheroma of human is a representative application applied to cardiovascular lesions, making sure the discrepancy between the product of gelatinases and their actual enzymatic activity is demonstrated by double stain of FIZ [23].
16.4 TECHNICAL HINTS AND DISCUSSION 16.4.1 Incubation Time of Film In Situ Zymography The optimal incubation time of FIZ depends on the purpose for which the staining is to be used. Different incubation periods are required to observe various lesions of the proteolysis properly [27]. For example, different incubation times are needed for the front line or whole mass of tumoral lesions and scattered cells, individually demonstrating gelatinolysis in the fibrous cap of atheromatous plaques. In the author’s experience, a maximum 6 hr of incubation usually provides an adequate result. And at a minimum, some cases showed their proteolysis with relative intensity and location correctly with only 30 min of incubation. It is this author’s hope that, in the midst of the complicated and time-consuming tasks of laboratory medicine in the hospi-
Figure 16.1 Double stain of immunogen and background gelatin. Double staining of HHF35 (black) and backgroundgelatin (red) distinguished area of high activity of gelatinolysis of some foam cells originating from smooth muscle cells from other foam cells, namely, macrophages in the atheromatous plaque of the human aorta of an autopsied case by sequential IGSS and Ponceau 3R procedures. (Original magnification sized in 14.5 × 10 cm, 40×.)
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Proteolytic Activity Demonstrated by Film In Situ Zymography (FIZ) tal, not only persons proficient in observing the morphology of human tissue but also any medical engineer or technician might be able to evaluate the activity of proteolysis using the fresh materials obtained from patients, including surgically resected specimens for diagnosis within 30 min. In the case of necessity of telling what kind of cell with immunocytochemical character should have equipped in the same location of their gelatinolysis, of course, IGSS on the slide is applicable for identification. 16.4.2 Comprehensive Analysis of Gelatinolytic Activity When it is possible to obtain large amounts of tissue samples, or when the sample requires only a short incubation time, it is possible to pre-estimate the comprehensive analysis of gelatinolytic activity. The combined methods of gel and ISZ checked its localization by IGSS would thus enable a decision as to optimal drug regimens for individual patients. It is possible to obtain sufficient sample by biopsy, cardiovascular intervention, or surgical resection from a patient to perform Mon
Tue
Wed
the method suggested herein. The combination of gel zymography [28], of conditioned medium and FIZ, on sections derived from the same tissue sample, provides information for the next step of treatment to avoid more proteolysis of tissue near the locus where the sample was obtained. The most preferable drugs would show minimal proteolysis in both methods as one of their pleiotrophic effects. This can be achieved in parallel in only one time course of combined analysis of gelatinolysis (Figure 16.2). 16.4.3 Estimation of Inhibition Effects in Film In Situ Zymography It is necessary to ascertain whether the proteolysis shown on the section is derived from the activity of MMPs or from other sources of proteases. Calcium is the most well-known inhibitor of MMPs, and not of other groups of proteases. For an estimation of the effect of inhibition by reagents to the tissue section on the glass slide, the dissolved solution of inhibitor can be used, mounted over the section. Even in the case of negative results, one cannot definitely say whether the effect is due to inhibition, or only due to the proteases released from
Thu
Fri
Sat
Sun
Cryosection Operation
Medical Decision
CM
Fixed material Figure 16.2 Time course of combined analysis of gelatinolysis. In the case of operation on Monday, after obtaining the surgical tissue materials, the author performed the dual zymographies in parallel and could finally report the results to the bedside within the same week. CM refers to conditioned culture medium.
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Molecular Morphology in Human Tissues: Techniques and Applications the tissue on the surface of the supportive slide, which has been diluted and not detected as proteolysis. Therefore, it is suggested that a piece of tissue should be immersed in inhibitor solution prior to freezing in OCT compound to ensure the real performance of their inhibitory effect: Tissue incubation with 20 mM EDTA for 5 min or with protease inhibitor added to conditioned media allows an estimation of their effects as an inhibitor. To achieve this under more controlled conditions, it is now possible to obtain a readily made FIZ gelatin film containing MMP inhibitor (1,10phenanthroline) commercially (“MMP-PT in situ Zymo-Film,” Cat. No. 291-58101, WAKO/Fujifilm, Wako Chemicals, Richmond, VA; http://home. fujifilm.com/products/science/techinfo/mmp.html). 16.4.4
Immunohistochemistry
The author has experimented with nine different monoclonal antibodies (CD 34, Factor VIII, actin, four MMPs, two TIMPs) and two polyclonal antibodies (three different antibodies to nitric oxide synthases) for immunohistochemistry after FIZ. Not all of the primary antibodies tested worked well in this particular setup. All of these antibodies did usually work on cryostat sections. Negative results, when combined with FIZ, are likely due to antigen masking and blocking caused by the gelatin substrate during the incubation process. To resolve these issues, the following three notations may be helpful: 1. 2.
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Disturbance of the connection between primary antibody and epitope can occur. Disturbance of infiltration of colloidal gold detecting the location of primary antibody/antigen complexes can occur.
3.
Incomplete silver enhancement can occur.
The above may be reasons for failing to detect the antigen in question. Heat antigen retrieval must be avoided here, also because of the character of gelatin substrate. Therefore, when (2) is considered, it would be worth trying new technologies, including Nanogold (Nanoprobes). Acknowledgments
The author is grateful to Dr. Gerhard W. Hacker for encouraging him to step into the new technology. This work was supported by a grant to promote research from Kanazawa Medical University (S2000-5) and a Grant-in-Aid (13770100) from the Japanese Ministry of Education, Science and Culture of Japan.
References 1.
2.
3.
4.
5.
Sappino, A.P. et al., Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney, J. Clin. Invest., 87, 962, 1991. Danscher, G., Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy, Histochemistry, 71, 1, 1981. Galis, Z.S., Sukhova, G.K., and Libby, P., Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques, J. Clin. Invest., 94, 2493, 1994. Galis, Z.S., Sukhova, G.K., and Libby, P., Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue, FASEB J., 9, 974, 1995. Curry, T.E., Song, L., Jr., and Wheeler, S.E., Cellular localization of gelatinases and tissue inhibitors of metalloproteinases during follicular growth, ovulation, and early luteal formation in the rat, Biol. Reprod., 65, 855, 2001.
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Proteolytic Activity Demonstrated by Film In Situ Zymography (FIZ) 6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Duchossoy, Y., Arnaud, S., and Feldblum, S., Matrix metalloproteinases: potential therapeutic target in spinal cord injury, Clin. Chem. Lab. Med., 39, 362, 2001. Freemont, A.J. et al., In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage, Ann. Rheum. Dis., 58, 357, 1999. Furuya, M. et al., Clarification of the active gelatinolytic sites in human ovarian neoplasms using in situ zymography, Hum. Pathol., 32, 163, 2001. George, S.J. et al., Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins, Gene Ther., 5, 1552, 1998. George, S.J. et al., Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein, Hum. Gene Ther., 9, 867, 1998. Goodall, S. et al., Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms, Circulation, 104, 304, 2001. Johnson, J.L. et al., Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques, Arterioscler. Thromb. Vasc. Biol., 18, 1707, 1998. Johnson, J.L. et al., Injury induces dedifferentiation of smooth muscle cells and increased matrixdegrading metalloproteinase activity in human saphenous vein, Arterioscler. Thromb. Vasc. Biol., 21, 1146, 2001. Lengyel, E. et al., Expression of latent matrix metalloproteinase 9 (MMP-9) predicts survival in advanced ovarian cancer, Gynecol. Oncol., 82, 291, 2001. Mungall, B.A. and Pollitt, C.C., In situ zymography: topographical considerations, J. Biochem. Biophys. Meth., 47, 169, 2001. Ikeda, M. et al., Inhibition of gelatinolytic activity in tumor tissues by synthetic matrix metalloproteinase inhibitor: application of film in situ zymography, Clin. Cancer Res., 6, 3290, 2000. Iwata, H. et al., Localization of gelatinolytic activity can be detected in breast cancer tissues by film in situ zymography, Breast Cancer, 8, 111, 2001. Takano, S. et al., Localization of gelatinase activities in glioma tissues by film in situ zymography, Brain Tumor Pathol., 18, 145, 2001.
19. Teesalu, T., Hinkkanen, A.E., and Vaheri, A., Coordinated induction of extracellular proteolysis systems during experimental autoimmune encephalomyelitis in mice, Am. J. Pathol., 159, 2227, 2001. 20. Yan, S.J. and Blomme, E.A.G., In situ zymography: a molecular pathology technique to localize endogenous protease activity in tissue sections, Vet. Pathol., 40, 227, 2003. 21. Zhang, J. and Salamonsen, L.A., In vivo evidence for active matrix metalloproteinases in human endometrium supports their role in tissue breakdown at menstruation, J. Clin. Endocrinol. Metab., 87, 2346, 2002. 22. Zheng, K. et al., A quantitative evaluation of active gelatinolytic sites in uterine endometrioid adenocarcinoma using film in situ zymography: association of stronger gelatinolysis with myometrial invasion, Jpn. J. Cancer Res., 93, 516, 2002. 23. Zuka M. et al., Vascular tissue fragility assessed by a new double stain method, Appl. Immunohistochem. Mol. Morphol., 11, 78, 2003. 24. Danscher, G., Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy, Histochemistry, 71, 1, 1981. 25. Hacker, G.W. et al., Silver acetate autometallography: an alternative enhancement technique for immunogold-silver staining (IGSS) and silver amplification of gold, silver, mercury and zinc in tissues, J. Histotechnol., 11, 213, 1988. 26. Holgate, C.S. et al., Immunogold-silver staining: new method of immunostaining with enhanced sensitivity, J. Histochem. Cytochem., 31, 938, 1983. 27. Zuka, M., Tanaka, T., and Okada, Y., Predictive value of gelatinase in urine and analysis of activation mechanism of MMP-9 for tumor progression, in Proc. of the 2nd Int. Conference on Tumor Microenvironment — Progression, Therapy & Prevention, Witz, I.P., Ed., Monduzzi Editore, Bologna, Italy, 2002, 153. 28. Nakamura, H. et al., Enhanced production and activation of progelatinase A mediated by membrane-type 1, 2, and 3 matrix metalloproteinase in human papillary thyroid carcinomas, Cancer Res., 59, 467, 1999.
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Conscious Production and Purchase of Reagents for Molecular Morphology: Methodological, Ethical, and Legal Considerations Gerhard W. Hacker, Antoine F. Goetschel, and Günter Schwamberger
17.1 INTRODUCTION In recent years, animal welfare has become a greater issue for scientists working in biomedical research. It is not only a matter of public demand for natural scientists generally to be more ethically concerned; it should also be a serious concern of scientists involved in all fields. Not aware of all the aspects involved, natural scientists usually buy reagents without addressing the issue of under what circumstances they have been produced, or what alternatives exist, which may yield the same or even better results than “conventional” animal-derived reagents. Antibodies produced using animals bind with high specificity and affinity to a seemingly limitless variety of biomolecules. They have an important, essential role in clinical medicine and basic biomedical research. Molecular morphologists and immunohistochemists rely on the use of antibodies as specific reagents to immunodetect peptides, proteins, or other substances expressing antigenic binding sites. In hybridization methods, antibodies are 0-8493-1702-9/05/$0.00+$1.50 © 2005 by CRC Press
used to detect reporter molecules labeling the site of specific nucleic acid hybrids. Most other reagents used in molecular morphology appear to be less related to this problem area, but play a role in supportive methods also applied by histochemists, such as tissue or cell cultures where fetal calf serum is used, although alternatives could be successfully used in many cases. This chapter draws attention to alternative methods of reagent production, methods that give comparable — and in some instances even better results — than those achieved with conventional animal-produced reagents. Because the scope of this chapter differs from others in this book, we do not give exact working protocols, but refer to selected key publications that describe these methods in detail. Emphasis is placed on new approaches to antibody production. We also highlight areas of which scientists should be aware when studying the catalogs of various antibody suppliers. The objective is the availability of catalog information with specific descriptions of the manner in which the reagents are produced, and also to put pres-
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Molecular Morphology in Human Tissues: Techniques and Applications sure on the industry to declare all details of their reagents’ production. Our purpose is to avoid animal experiments wherever possible or to minimize discomfort, distress, and pain in the care and use of animals in biomedical research. As indicated in this chapter and references cited herein, we believe that consequent attention to the “3R principle” (replacement, reduction, and refinement) will certainly contribute toward increasing the “humaneness of animal experimentation.” 17.2 POLYCLONAL ANTIBODIES 17.2.1 Polyclonal Antibodies in Molecular Morphology Molecular morphologists and immunohistochemists use polyclonal antibodies (PCAs) to (1) specifically detect substances in tissue sections (immunohistochemistry), cytological preparations (immunocytochemistry), or on Western blots, as “primary antibody”; (2) detect in situ the location of specific antigen-antibody-complexes formed in immuno(histo/cyto)chemistry; or (3) show the location of the specific (reporter-molecule-labeled) hybrids formed by in situ hybridization or in situ PCR on sectioned tissue, in cytology, or on blots. PCAs are usually the reagent of choice for “secondary” antibodies or antibodies binding to locations of reporter molecules. Instead of “primary” antisera, monoclonal antibodies (MCAs) are increasingly used for this purpose. It can be foreseen that pools of different MCAs, all recognizing different epitopes of the same antigen (class), will replace PCAs in many of these applications because their mass production in bioreactors will become more cost effective. For the time being, however, PCAs are still widely used.
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17.2.2 The Challenge: Ethical Considerations on Immunization Protocols 17.2.2.1
Freund’s Adjuvant
In the great majority of cases, immunization of animals is carried out by injections of immunological adjuvants together with an immunogen, that is, the substance against which a specific antibody is to be raised. Adjuvants are a large group of substances that stimulate the immune system without acting as antigens themselves. However, when applied together with an immunogen, the immune response against the immunogen (antigen) is augmented as compared to application of the immunogen alone. Thus, adjuvants belong to the group of immune modulators, that is, substances that are able to change the immunological response and enhance antibody production both quantitatively and qualitatively. Since 1916, oil emulsions have been used as adjuvants [1]. Complete Freund’s adjuvant (CFA), a mixture of killed mycobacteria, mineral oil, and an emulgator (Arlacel A), is the most commonly applied adjuvant, although being disputed increasingly. Different compositions of FA are in use. The CFA is regarded as an effective means of increasing primary humoral antibody responses to injected immunogens. The “incomplete FA” does not contain mycobacteria and is mainly used for booster injections. Many scientists, however, are not aware that use of FA causes pain and discomfort lasting days or even weeks for the laboratory animal, due to excessive inflammation, induration, and/or even necrosis [2]. Undesirable and painful effects, such as large inflammatory lesions or necrosis, can sometimes be reduced by the use of more
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Conscious Production and Purchase of Reagents for Molecular Morphology appropriate routes of administration. Although some authors try to justify the continued use of FA by arguing that it would not cause any discomfort when small volumes are injected intradermally or subcutaneously at multiple sites [3], one of us [4] has repeatedly observed adverse reactions in rabbits even in such application strategies in which multiple small- and content-adapted volumes at different injection sites were used. A study comparing different ways of application in mice and rabbits documents the discomfort caused by different immunization protocols [5]. Intraperitoneal or intramuscular injection quite often resulted in severe pathological conditions, fever, and pain. Both in rabbits and mice, only minimal differences in antibody titers were observed between the routes under study. Concluding from their observations, the authors preferred the subcutaneous route of injection for the induction of PCAs in rabbits and mice when an adjuvant is applied [5]. Intraperitoneal application [6] or injection into the foot sole produced severe pain and suffering. Corpuscular antigens (e.g., washed cells) can be applied intravenously without adjuvant and will still result in strong and rapid immune responses [7].
use of another adjuvant known to produce less or no inflammatory response, instead of, or at least before, using FA. In many parts of Europe, there are already legal rules that make this point official: scientists must first use alternatives. Only for very good and specifically defined reasons can FA be applied. Also, to justify whether at all the use of animals is allowed depends on what species and number of animals are needed, what method of immunization is to be chosen, and whether the immunization is really necessary.
To reduce pain for the laboratory animals in cases where FA is being used, it is important to use medication for pain reduction. In addition to conventional drugs for pain relief, herbal extracts routinely prescribed in traditional Chinese medicine for treatment of pain can be successfully applied, such as “DuHuo” (radix Angelicae pubescentis) [8].
In deciding which adjuvants should be applied to laboratory animals, it seems appropriate to ask which such products are being used for human vaccines [13]. In that field, it is very important to obtain high responses, effectiveness, and compatibility. The knowledge gathered from this field can be adopted for immunizing laboratory animals [14]. The question of safety and side effects of adjuvants remains unsolved. In humans, side effects have been studied and recorded in detail; and apart from local reactions, fever, polyarthritis, and even the induction of tumors have been observed. Only a few substances are regarded as less problematic for human
The University of Nebraska Medical Center has published guidelines (available on the Internet) on how to eliminate or reduce to a minimum animal discomfort associated with the use of FA in research [9]. The most important suggestion is the
The choice of adjuvant, the injection site, the volume of inoculations, the time and number of booster immunizations, the species, the characteristics of antigen, and the technique and volume of blood sampling are carefully regulated in Germany [10]. In the Netherlands, a “Dutch Code of Practice for the Immunization of Laboratory Animals” exists, with excellent compliance and experiences [11]. The pain load level with FA is regarded as being marked to severe, but appears to differ from species to species [7, 12]. 17.2.2.2 Alternatives to Freund’s Adjuvant
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Molecular Morphology in Human Tissues: Techniques and Applications vaccines, mainly aluminum compounds (such as aluminum phosphate) or calcium phosphate. Remarkably, FA is not used for human vaccines — it is regarded as much too toxic and pro-inflammatory, because formation of abscesses, fever, chronic changes in organs with generalized granulomatosis, and the induction of autoimmune diseases have been described [15]. Thus, it is difficult to understand that the use of FA in animals is still allowed, at least in many countries. It is up to us scientists to work on and with alternatives. Numerous concerned scientists throughout the world have described such alternatives. A number of authors compared different adjuvants in mammalian species such as rabbit, mouse, or rat. Evidence for a variety of well-applicable alternatives to FA, resulting in equally high or even increased antibody titers and associated with considerably fewer side effects, has been obtained [16–23]. In general, oil-based adjuvants often lead to undesirable discomfort of the laboratory animals [19,20]. Apart from FA, other mixtures relying on oil also need to be used with reservation: RIBI [19] or TiterMax injection [20], similar to FA, frequently resulted in significant and occasionally severe pathological changes. Another study describes TiterMax-induced inflammatory responses as mild or transient compared to those induced by FA [14]. Leenaars and colleagues suggest a better solution. From experiments with different oil-based adjuvants in combination with weak immunogens (synthetic peptides, glycolipids, and some particulate antigens), they concluded that high specific antibody levels along with limited side effects can be obtained by subcutaneous injection of peptide combined with Montanide ISA50 or Specol as a replacement of FA [12, 20]. Specol is also favored for certain applications in human vaccine production [23]. 256
Liposomes, for example, containing muramyl tripeptide phosphatidyl ethanolamine (MMP-PE), gave equal or better efficacy than FA and do not show toxic side effects [18]. Another approach is the use of lipopepides as a replacement for FA, which in some cases even yielded higher antibody titers than FA and at the same time appeared to be devoid of the side effects associated with FA [24, 25]. Polymethylmethacrylate nanoparticles, in one study described as the best overall adjuvant, can induce very high titers of antibodies without toxic side effects. The same study suggested that it may sometimes be required to test different adjuvants to induce the necessary immune response against physically different antigens [26]. On the other hand, the suggestion of an ethylene-vinyl acetate co-polymer (EVAc) as an antigen delivery device in laboratory rabbits seems to be an undesirable approach. Although three of the four animals tested did not exhibit inflammation or systemic illness in response to the pellet, the fourth rabbit repeatedly developed abscesses at the implantation site [27]. This approach was further complicated by subcutaneous implementation of the pellet devices. The role of whole mycobacteria, as contained in the complete FA, was questioned in the mid-1970s, and certain hydrosoluble components from mycobacterial and other bacterial origins were shown to be free of many side effects [28]. 17.2.2.3 Polyclonal Antibodies Produced in Hens (IgY) Recently, production of antibodies by laying hens has been described [29, 30], and industry has suggested that these would be a valuable alternative to mammalian PCAs. In their advertisements, they
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Conscious Production and Purchase of Reagents for Molecular Morphology try to convince scientists and the public that antibodies taken from egg yolk (IgY) are an important step in the direction of improved animal welfare. Are IgY antibodies suitable for applications in molecular morphology? A careful “Yes, but” may be the answer, as a number of problems related to this type of antibody production remain unsolved. IgY is the functional equivalent of IgG in birds, reptiles, and amphibia, but many aspects of its biology are poorly understood [29]. Numerous applications of this new type of reagent are emerging [30]. Depending on the purpose for which IgY antibodies are being raised, some of the hens used can live in relative freedom. Many of them, however, must be held in a fully sterile environment, thereby living in extremely small cages with much smaller living space than their pitiable sisters from egg plantation farms, without natural daylight, and being fed with fully sterile, unnatural food for their entire life. It appears to be a similar situation to that of many rabbits, mice, and rats being used for PCA production. So, what might be the advantages that would ethically justify this? There is one aspect that does not have to be performed anymore: it is not necessary to take blood to obtain the antibodies. Instead, egg yolk does contain the PCAs, and large amounts can easily be extracted from yolk [31, 32]. In this context only, producing antibodies in chicken eggs can be understood as some progress for animal welfare. For immunizing hens, adjuvants must be used, and many of them give rise to undesirable side effects. Earlier studies relied on FA, and some authors still describe successful applications of FAmediated immunization experiments in chickens [33]. Some authors state that FA
is well tolerated and would not produce inflammatory reactions in chickens [32]. There are numerous attempts to replace FA by other, less harmful adjuvants [34, 35]. Under appropriate immunization schedules that strictly avoid intramuscular administration, IgY antibodies with very high titers and a strong binding capacity (avidity), comparable to rabbit hyperimmune sera, can be obtained with minimal adjuvant side effects [35]. In some instances, the antibody titers obtained were significantly higher when lipopeptides were used as adjuvant instead of FA [34]. As in mammals, lipopeptides can be applied as an effective adjuvant with fewer side effects, thereby contributing to better welfare for the experimental animals [35]. Lipid nanoparticles as stable biocompatible adjuvants for chicken have recently been described, and may possibly be used for mammals as well [37]. There appears to be (at least for certain applications) a well-working alternative to immunization via injection of immunogen/antigen: oral immunization by gavage of laying hens with human IgG, combined with a number of potential adjuvants [38]. Quantification of the resulting immunospecific IgY antibodies by ELISA suggests that invasive technique-related stress could be eliminated/reduced in PCA-producing animals, a promising finding with implications not only for hens, but also for mammalian species [38]. For more than a decade, some immunohistochemists have tested IgY antibodies; however, relatively few publications deal with this application in comparison to mammalian antibodies. In a study where chlamydiae were to be detected in routine sections from formalin-fixed and paraffinembedded tissues from several species, it was reported that, in comparison to an optimally performing MCA, vitelline 257
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Molecular Morphology in Human Tissues: Techniques and Applications immunoglobulins produced some unspecific reactions, especially in lung tissue sections. Because of the antigenic relationship between the vitelline antibodies and tissues of birds, IgY is not suitable for the detection of psittacosis on avian substrates when using an indirect technique of immunohistochemistry. Staining of other tissues (e.g., intestine or placenta) was of comparable quality to that obtained with the MCA [39]. Another study described immunofluorescence applications of IgY [40]. In sum, IgY antibodies appear to display a number of advantages for certain applications: birds produce antibodies against highly conserved mammalian proteins; the quantity of antigen needed for immunization is very low; long-lasting IgY titers are predominant; antibody purification is simple, inexpensive, and quick; and chicken antibodies are acid- and heat-resistant. And, in contrast to bleeding animals, collecting eggs is non-invasive [32]. In particular, the heatresistancy of IgY might lead to interesting upcoming applications in molecular morphology, for example, those related to heat antigen retrieval and/or to nonproblematic heat-accelerated incubation conditions [41]. However, as noted above, cross-reactivity with endogenous tissue antigens may limit the potential usefulness of these reagents.
and/or “general markers,” but can also be observed for the detection of specific antigens such as neuropeptides. A relatively new approach is to apply mixtures of two or more MCAs together as a pool, yielding a defined PCA mixture with greatly enhanced specificity that potentially reduces the nonspecific background staining observed with conventional PCAs. 17.3.2 Ethical Considerations Related to Monoclonal Antibodies 17.3.2.1 Adjuvants for MCA Production MCAs also rely on laboratory animals; and as for the production of PCAs, it appears necessary to use adjuvants in most cases. Comparable ethical considerations to those mentioned above for PCAs should be considered when deciding which adjuvant is to be applied. A number of references already given for PCAs also deal with mice or rats; and as already pointed out above, species differences are sometimes observed. An example of a valuable study successfully reducing distress for laboratory mice in this context is the manuscript published by Ferber and colleagues [42]. 17.3.2.2
17.3 MONOCLONAL ANTIBODIES 17.3.1 Monoclonal Antibodies in Molecular Morphology For immunohistochemistry, in recent years a strong trend to replace PCAs with MCAs for specific primary antibodies probes has emerged. In many applications, these can be used as a single reagent, that is, as a probe containing only one type of MCA, and produce far cleaner results than PCAs. This is especially true when applied to the detection of tissue tumor markers 258
Ascites vs. In Vitro Methods
The procedures for the production of MCAs, however, differ significantly from those used for PCAs. Here we concentrate on those aspects related to animal welfare and to the applicability for laboratory scale, rather than detailing the differences between producing PCAs and MCAs. Two main methods are still in use for MCA production: (1) in vivo production of the final antibody inside mice peritoneum (the “ascites method”) and (2) production outside of animals. Incubators in a private lab can be used for small amounts, and biore-
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Conscious Production and Purchase of Reagents for Molecular Morphology actors can be for large-scale productions. The ascites method involves the hybridoma cells being inserted into the living animal, and this method already appears to be banned by the majority of scientists because severe distress and pain for the animals are invariably involved. The second reason for avoiding the ascites method is an economic one: production in bioreactors on a large scale can be more cost effective, depending on the method used. Surprisingly, published data available on animal-welfare issues related to the ascites method are limited [43–47]. Many researchers consider the mouse ascites method inexpensive and easy to use. Other arguments in favor of the ascites method include fast production and high yields, high titer, and cost efficiency. The principal argument against the ascites method is that the animals used are routinely subjected to chronic pain and distress. When injected into a mouse, the hybridoma cells multiply and produce fluid (ascites) in the animal’s abdomen. This fluid, containing a high concentration of the antibody, is also at the heart of the controversy concerning MCA production. Ascites fluid accumulation is known to cause discomfort and pain in human patients, and animals used in the ascites method frequently exhibit a spectrum of clinical symptoms, including anorexia, anemia, dehydration, difficulty in walking, respiratory distress, circulatory shock, classical peritonitis, fever, and pain-related behavior [48–50]. In recent years, research into in vitro alternative methods has progressed, and these approaches have become increasingly less expensive and more efficient. Using in vitro techniques, it is now possible to yield much higher concentrations of MCAs than in the early years of this technique. Furthermore, cleaner MCAs of high titer can be
obtained using in vitro cell culture techniques. Inflammation can never be totally avoided with the ascites method, and this inflammation adds additional “unspecific” antibodies to the resulting product as well as undesirable cytokines and chemokines. Many large laboratories around the world have already switched entirely to in vitro production. Other places, unfortunately, still overlook or ignore this elegant possibility for replacing the use of laboratory animals. In the decades that followed the original discovery by George Koehler and Cesar Milstein in 1975, tens of millions of animals suffered and died despite the availability of more humane alternatives. The appropriateness of using the ascites method was increasingly questioned in Europe; and since the end of the 1980s, one government after another in European states prohibited the ascites-based production of MCAs, thereby causing serious difficulties within the biomedical research community. Despite initial academic resistance, Germany and Switzerland followed the Netherlands’ governmental recommendations and laws, and bans of the ascites method became reality in Sweden and the United Kingdom [51]. In 1998, the European Commission and the European Center for the Validation of Alternative Methods (ECVAM) published a statement in favor of the scientific acceptability and practical availability of in vitro methods for the production of MCAs [52]. In the United States, the National Institutes of Health recently announced that it expects researchers to use in vitro methods whenever possible — and to offer very strong justification if they do plan to use the ascites method [53]. The U.S. National Research Council published a 47-page report of guidelines for monoclonal antibody production, wherein animal welfare plays an important role [54]. However, the ascites method is not yet prohibited in the United States, whereas many larger European 259
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Molecular Morphology in Human Tissues: Techniques and Applications countries have already completely banned this method — and for good reasons. In Australia, the National Health and Medical Research Council has suggested similar guidelines, in accordance with the 3R principle [55]; and The Monash, Australia’s most internationalized university, has published clear guidelines on the Internet [56]. 17.3.2.3 In Vitro Methods for Monoclonal Antibody Production An increasing variety of non-ascites methods are now available for MCA production, both for large and small volumes. In vitro methods include hollow fiber bioreactors [57], modular minifermenters, standard tissue culture flasks, and gas-permeable tissue culture bags [58, 59]. MCAs have been produced in chicken eggs [60], and there is even a recent report on MCAs successfully produced from plants [61]. Some researchers work on human MCAs [62]. For lab production of MCAs on a small scale, a new generation of membrane-based cell culture devices especially designed for this purpose can be used [63]. In contrast to conventional perfusion, hollow fiber bioreactors, these devices contain two functionally different membranes: one ultrafiltration membrane for nutrient supply and one gas-permeable membrane for direct oxygenation of cells. The latest systems of this generation are static culture systems that are of moderate cost and are either better than, or at least equal to, the ascites approach in terms of quality and quantity of produced MCAs [63]. 17.3.3 What Does It Mean to the Molecular Morphologist? In contrast to large-scale industrial production, which to a large degree has already switched to in vitro methods, at universities and other research institutions the pro260
duction of MCAs on the laboratory scale (milligram range) is still carried out in part by the ascites method. MCAs produced by the ascites method are also still being sold by some companies specializing in immunohistochemistry, immunology, and biochemistry. However, for the outcome of staining results to be obtained by immunohisto/cytochemistry or molecular morphology, it no longer matters how the MCAs are produced. A more ethically conscious purchase of MCAs — by selecting those not produced by the ascites method — should therefore be possible now without any disadvantage for the molecular morphologist. Specialized websites can help one obtain more information on this matter (e.g., on academic core centers and commercial facilities, where to obtain nonascites-produced MCAs) [58]. 17.4 RECOMBINANT ANTIBODIES In recent years, recombinant antibody technology has emerged as a potential alternative to conventional animal-derived antibodies used as molecular tools in immunochemistry. Because it is beyond the scope of this chapter to discuss the technical aspects of recombinant antibody technology in any detail, the reader is referred to recent books by Breitling and Dübel [64] and Kay et al. [65] as well as several excellent websites (see [66] and links given therein) for a more comprehensive discussion of basic as well as methodological aspects of recombinant antibodies. Rather, the intent here is to briefly summarize the major advantages as well as the drawbacks of this new technology in comparison with conventional animal-derived antibodies. Among various attempts to produce recombinant antibodies derived from animal or human immunoglobulin (Ig) gene
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Conscious Production and Purchase of Reagents for Molecular Morphology sequences in microorganisms, selection and production of recombinant antibodies via phage-display [67, 68] has emerged as the most frequently employed technique. Thus, we primarily focus this discussion on these types of reagents, together with a short survey on a more recent and very interesting development: the production of recombinant antibodies in plants, referred to as “plantibodies” (for a recent review, see [69]). 17.4.1 Recombinant Antibodies Generated via Phage Display Basically, for the production of recombinant antibodies via phage display, the complementarity determining regions (CDR) of the heavy and light chains of Ig genes with additional framework sequences are linked together to yield a linear sequence encoding a single antigen-binding domain. This domain is further linked to various molecular tag structures that allow specific detection and simple purification of these so-called single-chain variable (Ig) fragment (scFv) structures. Using suitable linking sequences, these scFv can then be expressed either as fusion proteins to the gene-III coat protein of filamentous M13 phages or as soluble “secretory” proteins by E. coli. Large synthetic phageimid-encoded scFv libraries are generated by random permutation of CDR sequences, yielding theoretical complexities of up to 109 different clones. Selection of antigen-specific clones is achieved via binding of recombinant scFv-decorated phage particles to an immobilized antigen of interest, followed by desorption of bound phages and infection of suitable E. coli host cells to produce progeny of recovered phages for further selection. Using several of these so-called “panning” rounds, antigen-binding phages are amplified and phage clones specific for a defined antigen can be recovered and the
corresponding scFv expressed in E. coli for production of purified scFv recombinant antibodies. 17.4.2 Advantages of Phage DisplayBased Recombinant Antibody Technology Theoretically, this technique offers several advantages over conventional animalderived antibody production. First, and probably most important, the use of synthetic scFv libraries, derived from animal or human germline CDR sequences, allows for selection of recombinant antibodies to virtually any molecular structure, irrespective of its immunogenicity in conventional immunization procedures, because the scFv libraries are not subject to negative repertoire selection resulting in tolerance to self-antigens. Thus, the critical problem of low or totally lacking antigenicity of many relevant, highly conserved biological structures in animal immunization procedures is circumvented. Second, the actual amount of antigen required is very low (in the nanogram range for typical protein antigens) compared with conventional immunization protocols, which require at least microgram quantities of purified antigens. Third, generation of monoclonal recombinant antibodies via phage display can be achieved within a few weeks at fairly low cost, requiring only basic laboratory equipment and comparatively inexpensive media and reagents, and of course obviating the need for animal housing facilities. Fourth, selected clones offer a stable source for, at least theoretically, unlimited recombinant antibody production from laboratory to industrial scales. And finally, the use of defined tag structures allows for simple purification and highly specific detection of scFv used as molecular probes in immunostaining.
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Molecular Morphology in Human Tissues: Techniques and Applications 17.4.3 Drawbacks of Phage DisplayBased Recombinant Antibody Technology Given these advantages, it may seem surprising that this technology currently is not used to a larger extent, both by researchers and industrial companies. This may be partly due to limited awareness of these fairly novel techniques among researchers as well as “sticking to old habits,” but also reflects several drawbacks of recombinant antibody technology, which however can be circumvented at least partially by choosing suitable experimental conditions. The first and probably most crucial drawback of scFv is their usually considerably lower antigen affinity (20-fold or more), compared with conventional monoclonal or polyclonal antibodies. If used as molecular probes for immunostaining, this requires considerably larger amounts of scFv compared with normal antibodies for sensitive staining, and in turn also causes increased background staining due to nonspecific adsorption of scFv to irrelevant structures. However, the affinity of scFv can be enhanced to levels almost comparable with conventional MCAs by mutating the scFv coding sequences of already-selected phage clones and reselection of antigen binding phages under conditions of high stringency, thus mimicking somatic hypermutation and clonal selection of Ig genes during immune reactions in mammals. Although time consuming, this approach represents the best way to generate high-affinity recombinant antibodies for use as molecular probes. Alternatively, scFv-expressing phages can be directly used as reagents for immunostaining under certain circumstances. The advantage of these so-called “phage-bodies” as staining reagents stems from the fact that up to five identical scFv molecules can be displayed on the tip of a single phage particle, thus increasing the practical affin262
ity for the antigen [70]. In addition, the polymeric structure and large size of the phage particle (close to 1 µm in length) allows for multiple binding sites for phagespecific secondary antibodies or direct labeling of phage particles for highly sensitive detection of bound phages. In practice, the increase in sensitivity obtained is more than 100-fold [71], matching the sensitivity of conventional antibodies. Unfortunately, this can be associated with considerable nonspecific background binding due to the highly charged surface of phage particles, which may not be eliminated completely by the use of conventional blocking agents. Furthermore, the size of the phage particles limits the resolution of phagebody-mediated immunostaining to about 2 µm, which may be too low for certain applications in molecular biology. The second major drawback of phagedisplayed scFv is that in contrast to the expression as pIII-fusion proteins and the display on the phage surface, the large-scale production of soluble scFv as “secretory” molecules faces severe problems. Induction of scFv production in E. coli often results in the accumulation of misfolded, nonfunctional protein aggregates (so-called inclusion bodies) and the concomitant death of producer bacteria, which practically limits the output of functional scFv molecules to often considerably less than 1 mg/l of bacterial culture. Because this is largely dependent on the ultimate protein structures encoded by the selected clones, both output quantities as well as qualities of the produced scFv may vary considerably from clone to clone. Quite frequently, no functional soluble scFv can be obtained from a single selected antigen-binding phage clone. Therefore, in practice, several different phage clones specific for a certain antigen must be tested for production of functional soluble scFv. Numerous attempts have been made to overcome this
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Conscious Production and Purchase of Reagents for Molecular Morphology problem with single phage clones [64, 72, 73]; however, the general feasibility of these protocols remains to be established. Thus, the step from an antigen-specific phage clone to suitable amounts of functional soluble scFv can be quite tedious. Again, this problem can be overcome by the use of “phage bodies” as molecular reagents if applicable, given the restrictions discussed above. Thus, while the phage display system offers a very efficient and convenient way for selection of recombinant antibodies to almost any molecular target structure, equally efficient and reliable techniques for large-scale production of these antibodies are still lagging behind, which to date represents the major limitation of recombinant vs. conventional antibody technology. Nevertheless, and despite all these limitations, phage bodies and functional soluble scFv have been successfully produced to a variety of molecular target structures [74, 75], thus further underlining the practical potential of this technique for generating molecular tools for immunochemical methods. Given the development of improved methods for large-scale bioproduction of recombinant antibodies, this technique may gradually replace conventional immunization and antibody production procedures. 17.4.4 Antibodies Produced in Plants In comparison with bacteria, plant cells offer a much more suitable chemical microenvironment for the synthesis, proper folding, and posttranslational modification of recombinant proteins in general. This also holds true for recombinant antibodies and so, to date, a variety of both cloned natural, animal-derived or human Ig-molecules, and fragments as well as recombinant scFv generated via phage display have been successfully produced in
crop plants and have been used for diagnostic and therapeutic purposes (for review, see [69]). The main advantage of antibodies produced in plants is the comparatively high yield of functional antibody molecules, which, after purification, can be directly used as a substitute for conventional antibodies, obtained from laboratory animals or animal cell culture. The second advantage is the low production cost once the recombinant plants have been generated. This latter point, however, also represents the most serious drawback of plantibodies, because the generation of recombinant plants, the growth of the crop, and the purification of the antibody is a rather complex, time-consuming task, estimated to require about 2 years to set up large-scale production [69]. For this reason, the effort is probably too great for research laboratory-scale production, and thus this technology will quite likely remain restricted to pharmaceutical and biotech companies. In additon to these economic considerations, concerns have been raised about the potential hazards of uncontrolled biodistribution of genes encoding antibodies to structures relevant to human health. This demands production of recombinant plants under containment conditions, thereby markedly increasing production costs. Nevertheless, it appears likely that within the next decade, both natural as well as recombinant antibodies produced in plants will gradually replace commercially available, conventional antibodies. Taken together, recombinant antibody technology, although methodically not yet fully mature, undoubtedly offers a promising alternative to the conventional generation of diagnostic and therapeutic molecular tools in the near future and thus hopefully will reduce the amount of animal experimentation still required in biomedical research. 263
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Molecular Morphology in Human Tissues: Techniques and Applications 17.5 FETAL CALF SERUM Fetal calf serum (FCS), although not a reagent for immuno(histo/cyto)chemistry itself, is widely used as a supplement considered to be essential for animal cell culture media and thus is also used for the in vitro production of MCAs. However, the use of FCS as a standard medium additive for cell cultivation must be regarded critically, both from the point of view of animal welfare and for scientific reasons, arguing for alternatives [76–78]. While the use of 10% FCS as a media supplement may indeed constitute an essential source of hormones for culture of primary cells, it is rarely essential for the growth of established cell lines. Thus, although several expensive and proprietary commercial substitutes for FCS exist (refer to supplier catalogs for information), in the experience of one of the authors [71], most established cell lines can be grown in a rather simple and inexpensive medium consisting of 50% Iscove’s modified Dulbecco’s medium and 50% HAM’s F12 medium, supplemented with bovine insulin (2.5 µg/ml), bovine holo-transferrin (5 µg/ml) and bovine thyroglobulin (5 µg/ml), provided that the cells are adapted to these conditions slowly. In practice, conventional FCS-supplemented cell cultures can be adapted to serum-free growth conditions by daily twofold dilution of the FCS-supplemented, used medium with the fresh serum-free medium described above, while trying to keep the cell density at about 2–5 × 105 cells/ml. Typically, at a residual FCS concentration of about 0.5%, growth rates decrease somewhat and further splitting should be slowed down as well to allow the cells to overcome this hormonally critical phase. Altogether, under these circumstances, most established cell lines can be adapted to serum-free culture conditions, resuming normal growth characteristics within about 2 weeks; whereas for those 264
few lines that cannot be adapted successfully, 0.5 to 1% FCS is usually sufficient to sustain normal growth. In the experience of one of the authors [71], this scheme also works well with most hybridoma cell lines used for MCA production. The only caveat associated with serum-free culture conditions is that cultures should not be split to densities below about 2–5 × 104 cells/ml, because these cultures usually depend on autocrine growth factor production. Apart from the markedly reduced costs, serumfree cell culture offers several advantages over conventional FCS-supplemented cultures, such as the reduced risk of contamination with viruses and mycoplasms and the highly standardized conditions, compared with the considerable batch-to-batch variability of FCS sources. From a technical viewpoint, however, the most important advantage is the markedly simplified processing of conditioned culture media for purification of secretory products such as MCAs, which is not hampered by the presence of vast amounts of serum components. Thus, also from a methodological point of view, it seems worthwhile to consider serum-free cell culture as an alternative to conventional FCS-supplemented cell culture whenever possible. 17.6 LEGAL ASPECTS 17.6.1 General Legal Aspects Animal welfare laws protect animals against pain, distress, and suffering caused by humans. Usually they distinguish various sectors, depending on the use of animals in man’s responsibility: companion animals, farm animals, wild animals, and laboratory animals. The human–animal relationship is based on ethical and religious opinions. How far can man go in using — or abusing — animals for his own purposes [79, 80]? The answer is often
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Conscious Production and Purchase of Reagents for Molecular Morphology given on a personal basis, depending of one’s own education, work, and familiar surroundings. In the legislative and implementation process, ethical guidelines play an important role. They are often used as a lighthouse, giving a clear direction in which public opinion and the law should go. It makes a big difference whether, for example, the “dignity of animals” is protected by a constitution, as in Switzerland since 1992, or only vertebrates except rats and mice are protected against pain. Thus, the differences among the various legislations within the field of laboratory animals are quite obvious and can only be pointed out very briefly within this chapter. U.S. legislation, as well as some examples valid for the European Union, Germany, Austria, and Switzerland, are discussed below. 17.6.2 Legal Aspects in the United States In the United States, the Federal Animal Welfare Act (AWA) (7 U.S.C. 2131-2157) is the first and only major federal legislation governing the use of animals in research, education, and testing. It contains the primary legislation enacted governing the use of animals in biomedical experimentation. The Act is dated 1970 and applies to any research facility. It includes provisions for the administration of painrelieving drugs, minimum-size requirements for holding cages, and the establishment of institutional review boards to minimize or prevent duplication of experiments and to examine their protocols [81]. Different amendments were enacted (in 1976, 1985, and 1990). In 1985, the Improved Standards for Laboratory Animals Act was enacted into law. It strengthened care standards, increased enforcement, required training of animal handlers and relief of pain and distress. These
amendments necessitated the establishment of an information service in the National Agriculture Library in cooperation with the National Library of Medicine. The service maintains data that assists in preventing unintended duplication of experiments and tests, in finding alternatives to the use of laboratory animals in experiments, and in instructing scientists and laboratory employees concerning the humane animal practices now required under the law [82]. Consultation with a veterinarian is also necessary for designing research with painful procedures, and for supplying adequate pre- and post-surgical care [83]. To ensure compliance with these new rules, every research facility is required to establish an Institutional Animal Care and Use Committee (IACUC) composed of at least three members. One person must represent “general community interests in the proper care and treatment of animals.” A voting majority of members is necessary to approve any committee actions or resolutions. The IACUC is charged with the responsibility of reviewing all research proposals using animals, ensuring that investigators will provide appropriate pain relief and euthanasia and have considered alternatives to using animals [83]. Inspections are overseen, and a report of the findings of the IACUC must be filed with the Animal and Plant Health Inspection Service (APHIS). Additionally, all federally funded research must comply with the “Public Health Service Policy on Humane Care and Use of Laboratory Animals” and the “National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals” [83]. As far as rabbits and goats are concerned, animals frequently used in the production of antibodies, the Improved Standards for Laboratory Animals Act requires, 265
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Molecular Morphology in Human Tissues: Techniques and Applications for example, consultation with a veterinarian on possible pain reduction. Viable alternatives to the experiments are considered, and the people involved with animals are trained in humane animal maintenance and experimentation, and the reduction of the number of animals used and the pain caused to animals. Undoubtedly, the Federal Animal Welfare Act brought about improved care of animals in experiments. Nevertheless, comparing the elaborate system of legislation to European standards, several aspects must be looked at more closely. First, the regulations subsequently issued by APHIS under the authority of the AWA define “animal” to mean “any live or dead dog, cat, monkey (non-human primate mammal), guinea pig, hamster, rabbit or such other warm-blooded mammal, as the Secretary may determine is being used … for … experimentation.” In 1972, APHIS issued a regulatory definition of “animal” stipulation that “this term excludes: birds, rats and mice bred for use in research.” However, the latter two species alone account for approximately 90% of all laboratory animals, yet they are not at all protected by the AWA or its regulations and are not taken care of in the statistics. Estimates vary from 10 million up to 25 or even 70 million per year compared to 2.21 million other animals in experiments in 1998. The IACUC is not authorized to promulgate rules or orders with regard to the performance of actual research or experimentation by a research facility, as determined by such research facility [82, 84] (7 U.S.C., § 2143 (a)(6)(A)(ii)). The Act permits the animals to be used for any purpose that is deemed “necessary” to the experiment. 266
Third, the AWA does not oversee members of the IACUC nor animal welfare rights organizations to appeal a decision made by the majority of the IACUC members and to go to court. IACUC members are not authorized to engage in any ethical review (7 U.S.C., § 2143 (b)(1)). Thus, ethical aspects — on the issue of whether the experiment is necessary to achieve a certain goal or if the goal by itself is serious enough to be worth an animal’s suffering and killing — cannot be discussed, neither by the IACUC nor by a judge in applying the AWA or the 1985 amendment. 17.6.3 Legal Aspects in the European Union Legislation in the European Union only partially captures experimental animal research. Binding standards can be found in various legal records of the community legislation, and in particular in the directive for animal experimentation 86/609/EEC. This directive, however, does not represent an actual animal protection measure but rather one of harmonization, with the primary goal of unification of the regulations of the participating states in order to prevent distortions of competition and trade barriers that could harm the Common Market. Although the directive contains some practicable approaches in the direction of an up-to-date animal experimentation law, it only defines general goals, which allow for considerable leeway in national implementation within the individual EU countries, and it has only a limited area of legal operation [85, 86]. On the one hand, only vertebrates are included; and on the other, the law focuses on applied research and protects only animals used in product and substance development or test procedures as well as those used in the framework of environmental protection. Various important fields of
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Conscious Production and Purchase of Reagents for Molecular Morphology research are thus not subject to a common regulation and are assigned to national legislation. This concerns animal experimentation in education and training or for military or so-called defense-relevant medical purposes and, in particular, the entire area of basic research, including the field of genetic engineering in animals with its growing significance. The EU directive 86/609/EEC is almost identical to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes of the Council of Europe (ETS 123) dated March 18, 1986, and entered into force in 1991. The Convention is binding for European states that are not part of the EU (as, for example, Switzerland) that ratified it. The analysis of the EU directive can be easily extended to the Convention of the Council of Europe. One of the most important focuses of the EU directive is the reduction of an animal’s pain and suffering due to experiments. For example, an animal shall not be used for more than one time in any experiments entailing severe pain, distress, or equivalent suffering (Article 10). “Where it is planned to subject an animal to an experiment in which it will, or may, experience severe pain which is likely to be prolonged, that experiment must be specifically declared and justified to, or specifically authorized by the authority.” The authority shall take “appropriate judicial or administrative action if it is not satisfied that the experiment is of sufficient importance for meeting the essential needs of man or animal” (Article 12). A basic distinction between European and U.S. animal welfare laws illustrated by this rule is noted. On the one hand, an ethical decision must be made in weighing the animal’s suffering against essential needs of man or animal. On the other hand, the authority must
decide on an experiment — not merely the scientist himself or a private committee. Similar to the AWA, the EU directive obliges member states to ensure that all experimental animals shall be provided with housing, an environment, at least some freedom of movement and care, that the well-being and state of health must be observed to prevent pain or avoidable suffering, distress or lasting harm, and that arrangements are made to ensure that any defect or suffering discovered is eliminated as quickly as possible (Article 5). All vertebrates are protected, as well as rats, mice, and birds. Experiments shall be performed solely by competent authorized persons, or under the direct responsibility of such a person, or if the experimental or other scientific project concerned is authorized in accordance with the provisions of national legislation (Article 7(2)). An experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available (Article 7(2)). From Article 7(2), the immunization need not be performed by a veterinarian, medical doctor, or biologist, but may also be carried out by other persons, under his or her direct responsibility, who are authorized by the legislation of the EU member state. Some existing production and testing methods for antibodies from animals are scientifically unsatisfactory, and may cause unnecessary pain and suffering and demand large numbers of animals. Consideration should be given to alternative methods. The EU member states are explicitly entitled to apply or adopt stricter measures for the protection of animals used in experiments or for the control and restriction of 267
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Molecular Morphology in Human Tissues: Techniques and Applications the use of animals for experiments. In particular, member states may require a prior authorization for experiments or programs of work notified in accordance with the provisions of Article 12(1). As examples, Germany and Austria will be looked at more closely with their recently revised animal welfare acts, as will Switzerland as a non-EU member state [85]. 17.6.4 Legal Aspects in Germany The Animal Welfare Act of Germany is dated May 25, 1998/August 6, 2002, and regulates animal experiments in § 7 to § 10a. It mandates a fine-tuned system of authorization or indication for animal experiments, for the use of animals in education and training (§ 10) and in the production of tissues and organisms (§ 10a). Animal experiments must be ethically defendable; that is, the expected harm to animals must be in reasonable relation to the goal of the experiment (§ 9 (3)). Experiments on animals are to be restricted to the “absolutely essential degree” (unerlaessliches Mass; § 9 (2)). The public authority licenses an experiment with vertebrates if the legal conditions are fulfilled. A commission with scientists and animal welfare specialists work out suggestions to the state authority, and determines whether the planned experiment must be stopped or under conditions it can be permitted (§ 15 (1)). Often, there is no consensus within this commission, but the suggestion is not legally binding [87]. In practice, the use of mice with ascites for the production of monoclonal antibodies is forbidden, except for emergency cases in diagnostics or therapy of man, the “saving” of hybridomas when they are not growing any longer in cell cultures or when infected, or for working out new scientific questions [87–89]. Such production is illegal and can be punished under § 17 (2b) 268
or § 18 (1)(1) of the Animal Welfare Act, because the antibodies can easily be produced with durable cell lines in vitro [87, 89]. Basically, anesthesia must be administered either by a specialized person (e.g., a veterinarian or medical doctor) or someone under his or her supervision (§ 9 (2)(1)). 17.6.5 Legal Aspects in Austria The Animal Welfare Law in Austria protects animals against pain and suffering. The field of animal experiments is part of the union state level. The Austrian Animal Experiment Law, dated September 9, 1989, has as its goal to replace and refine animal experiments, and to reduce animal experiments to the necessary minimum (§ 1). Only vertebrates are protected, and every animal experiment requires that a permit be issued by the state authority (§ 5–10), be it the Central Ministry of Science and Research (BMWF) or the head of the provincial government (“Landeshauptmann”) [85]. Permits cannot be taken to court, neither by members of commissions nor by animal welfare organizations. Raising of antibodies is allowed explicitly, unless accepted alternative methods exist [90]. Larger refinements of the Animal Welfare Law, also toward responsibility of the central state, are currently in progress. Austria is a member of the EU and must implement the 86/609 EEC directive. 17.6.6 Legal Aspects in Switzerland In Switzerland, animal experiments require authorization and must be restricted to the “degree absolutely essential” [91]. The provisions for animal experiments are found in Articles 12 to 19b of the Animal Welfare Act dated March 9, 1978 (July 1 1995 version); Articles 58 to 64b of the Animal Welfare Regulation (dated of May 27, 1981 [October 27, 1998 version]); and
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Conscious Production and Purchase of Reagents for Molecular Morphology in directives and guidelines issued by the Federal Veterinary Office [92]. Similar to the German legislation, a commission on the level of the cantons (states) works out suggestions on planned experiments with vertebrates that may cause harm to animals. The state authority licenses the experiment if it is in compliance with the legal framework. As a reaction to a people’s initiative, the Animal Welfare Act was revised in 1991 and the Swiss Federal Veterinary Office is entitled to make an appeal against the permit (§ 26a). To enforce the animal’s interest, the Animal Welfare Act of the Canton of Zurich was revised in 1991; this revision allows the commission, as well as a minority of this commission, to appeal the granting of a license by taking it to the court for administrative law. Because three members of the commission must be designated by animal welfare organizations, they have the right to defend animal rights in court. In addition, in the Swiss canton of Zurich, an office for an “attorney for animal welfare in criminal cases” was created in 1991, to our knowledge the only such office in the entire world. The animals’ attorney represents the aggrieved vertebrate animal in every criminal case involving cruelty to animals perpetrated in the canton of Zurich [93]. Thus, vertebrate animals have standing, at least within this small range of criminal cases and in going to court against illegal permits for animal experiments in the canton of Zurich. Switzerland is, to our knowledge, the only country to have the “dignity of animals” protected in its constitution. The need for a fundamental change in our relationship with animals gave rise to the idea of granting animals “dignity” on the highest legal basis, as added to the Swiss constitution in 1992. Thereafter, animals are not only protected against suffering, pain, distress, and fear, but also in natural integrity,
that is upheld as long as it can retain its independent viability, its “otherness” as an animal, its specific “such-ness,” and its possibilities for development despite being exploited and bred by humans. The “dignity of animals” has not yet been incorporated into the Animal Welfare Act of 1978, although revision work is ongoing. The Swiss Veterinary Office has worked out several guidelines to facilitate the application of the animal experiment law in the licensing system. The information on the categories of animal experiments by strain before the start of the experiment (Nr. 116.104) addresses the second grade (of three) for the subcutaneous or intramuscular immunization of rabbits, mice, rats, and guinea pigs using the Freund’s or similar adjuvant. Every immunization of an animal with tissues derived from its own body, thereby leading to an autoimmune illness if the experiment has not been stopped in time, is classified as a “very straining” experiment (highest grade 3). The 5.01 guideline of the Swiss Veterinary Office on the production of monoclonal antibodies describes this method of using animals, in principle, as unnecessary. Monoclonal antibodies can be produced in vitro by cell cultures. As an exemption, these alternative methods do not lead to the success expected. Similar to the German practice, applications for the ascites procedure can be licensed in emergency cases in human medicine (e.g., in the treatment of individual patients) or for the preservation of hybridoma cells if it is documented that they cannot or do not sufficiently grow, or are infected. A 1999 Swiss Veterinary Office guideline on professional and animal welfare addressed production of antibodies in rabbits, chicken, mice, and rats. Therein, it is recognized that the production can easily produce significant strain on the animals, especially because of 269
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Molecular Morphology in Human Tissues: Techniques and Applications infections, and guideline shows several ways of refining the methods [94]. 17.7 CONCLUDING REMARKS The lack of awareness, limited time resources, and pressure for success are among the main reasons that many scientists often do not really care about how the reagents they are using have been produced. We believe that it would be huge progress for the natural science community if we could start to concentrate on a more holistic understanding of our work and or our being on earth. Signs of increased awareness in this field already exist, as evidenced by an enormous number of research articles and Internet-based discussions dealing with this question. As can be seen from the overall number of publications cited in a recent “Antibody Production Bibliography,” the concern about ethical questions is rapidly growing [95]. Most reagent supplier catalogs, however, do not provide any detailed information on this topic, for example, if an MCA derives from the ascites method or if FA has been used for immunization. This chapter has been written not only as an aid to increase the ethical awareness of the “user,” but it also aims to request suppliers to make this information visible at first sight inside their catalogs and product descriptions. Examples of companies applying gentle immunization procedures only, in close concordance with animal welfare ethics, already exist (e.g., ImmunoGlobe) [96]. What can the molecular morphologist or immunohistochemist contribute to this situation? Only very recently has a trend in our field toward a more ethically concerned way of thinking been observed. Prominent histochemistry journals (such as the Journal of Histochemistry and Cytochemistry) have recently added questions on ani270
mal care to their guidelines to authors and to their referees’ questionnaires. Buying reagents, or self-producing them, should be understood as an integrative part of this question. We can ask antibody suppliers for more details on how the MCA or PCA was produced. If the ascites method or FA has been used, we should consider not buying such antibodies and look for alternatives. We can also put pressure on distributors. As a matter of fact, this increasing demand will certainly lead manufacturers to take these concerns into account, and bring new products to the market that take animal welfare issues into account. With time, many of them will remove “old” products from their shelves and catalogs, and those companies that act first may even gain economic profit from such decisions. In addition, improved awareness on the part of molecular morphologists regarding ethical aspects related to antibodies will definitely help stimulate further research and new approaches in this field. Thus finally, with this chapter we hope to have raised an increased awareness of animal welfare aspects involved in the production of antibodies and at the same time to provide some methodological clues for alternative techniques for the benefit of laboratory animals.
References 1.
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Hilleman, M.R., Critical appraisal of emulsified oil adjuvants applied to viral vaccines, Prog. Med. Virol., 8, 131, 1966. Deeb, B.J. et al., Comparison of Freund’s and Ribi adjuvants for inducing antibodies to the synthetic antigen (TG)-AL in rabbits, J. Immunol. Meth., 152, 105, 1992. Halliday, L.C. et al., Physiologic and behavioral assessment of rabbits immunized with Freund’s complete adjuvant, Contemp. Top. Lab. Anim. Sci., 39, 8, 2000. Hacker, G.W., unpublished data, 1985–1990.
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Leenaars, M. and Hendriksen, C.F., Influence of route of injection on efficacy and side effects of immunisation, Altex, 15, 87, 1998. Toth, L.A. et al., An evaluation of distress following intraperitoneal immunization with Freund’s adjuvant in mice, Lab. Anim. Sci., 39, 122, 1989. Kuhlmann, I.I., Storz, B., and Gast, I., Erfahrungen mit dem Einsatz des alternativen Ribi-Adjuvans-Systems zur Gewinnung von Antiseren zu Forschungszwecken. (Experiences on the use of the alternative Ribi-adjuvant for raising antisera for research purposes), Der Tierschutzbeauftragte, 2, 136, 1995. Wei, F. et al., Effects of four herbal extracts on adjuvant-induced inflammation and hyperalgesia in rats, J. Altern. Complement. Med., 5, 429, 1999. University of Nebraska Medical Center, Guidelines for Use of Freund’s Adjuvant in Laboratory Animals, http://www.unmc.edu/Education/Animal/ guide/appenJ2.html. Landwehr, M., Die tierschutzrechtliche Beurteilung der Immunisierung von Tieren in Deutschland, in Ersatz- und Ergänzungsmethoden zu Tierversuchen, Schöffl, H., Spielmann, H., and Tritthart, H.A., Eds., Springer, Vienna/New York, 1996, 204. De Leeuw, W.A. and De Greeve, P., Experience with the Dutch code of practice for the immunization of laboratory animals, in Ersatz- und Ergänzungsmethoden zu Tierversuchen, Schöffl, H., Spielmann, H., and Tritthart, H.A., Eds., Springer, Vienna/New York, 1996, 210. Leenaars, P.P. et al., Assessment of side effects induced by injection of different adjuvant/antigen combinations in rabbits and mice, Lab. Anim., 32, 387, 1998. Allison, A.C., Adjuvants and immune enhancement, Int. J. Technol. Assess. Health Care, 10, 107, 1994. Bennett, B. et al., A comparison of commercially available adjuvants for use in research, J. Immunol. Meth., 153, 31, 1992. Gupta, R.K. et al., Adjuvants — a balance between toxicity and adjuvanticity, Vaccine, 11, 293, 1993. Claassen, E. and Boersma, W.J., Characteristics and practical use of new-generation adjuvants as an acceptable alternative to Freund’s complete adjuvant, Res. Immunol., 143, 475, 1992. Kellner, J. et al., The influence of various adjuvants on antibody synthesis following immunization with a hapten, Biol. Chem. Hoppe Seyler, 373, 51, 1992.
18. Ullrich, S.E. and Fidler, I.J., Liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) are excellent adjuvants for induction of an immune response to protein and tumor antigens, J. Leukoc. Biol., 52, 489, 1992. 19. Leenaars, P.P. et al., Evaluation of several adjuvants as alternatives to the use of Freund’s adjuvant in rabbits, Vet. Immunol. Immunopathol., 40, 225, 1994. 20. Leenaars, M. et al., Immune responses and side effects of five different oil-based adjuvants in mice, Vet. Immunol. Immunopathol., 61, 291, 1998. 21. Zwerger, C. et al., A comparison of commercially available adjuvants in BALB/c-mice immunised with a weekly immunogenic peptide, Altex, 15, 83, 1998. 22. Weeratna, R.D. et al., CpG DNA induces stronger immune responses with less toxicity than other adjuvants, Vaccine, 18, 1755, 2000. 23. Beck, I. et al., Investigation of several selected adjuvants regarding their efficacy and side effects for the production of a vaccine for parakeets to prevent a disease caused by a paramyxovirus type 3, Vaccine, 21, 1006, 2003. 24. Bessler, W.G. and Jung, G., Synthetic lipopeptides as novel adjuvants, Res. Immunol., 143, 548 and discussion 579, 1992. 25. Baier, W. et al., Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization, Immunobiology, 201, 391, 2000. 26. Stieneker, F. et al., Comparison of 24 different adjuvants for inactivated HIV-2 split whole virus as antigen in mice. Induction of titres of binding antibodies and toxicity of the formulations, Vaccine, 13, 45, 1995. 27. Niemi, S.M. et al., Evaluation of ethylene-vinyl acetate copolymer as a non-inflammatory alternative to Freund’s complete adjuvant in rabbits, Lab. Anim. Sci., 35, 609, 1985. 28. Jolles, P., Hydrosoluble immunostimulants of bacterial and synthetic origins, Experientia, 32, 677, 1976. 29. Warr, G.W., Magor, K.E., and Higgins, D.A., IgY: clues to the origins of modern antibodies, Immunol. Today, 16, 392, 1995. 30. Schade, R. and Hlinak, A., Egg yolk antibodies, state of the art and future prospects, Altex, 13, 5, 1996. 31. Polson, A., von Wechmar, M.B., and van Regenmortel, M.H., Isolation of viral IgY antibodies from yolks of immunized hens, Immunol. Commun., 9, 475, 1980.
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Molecular Morphology in Human Tissues: Techniques and Applications 32. Gassmann, M. et al., The chicken egg as a supply of polyclonal antibodies, Schweiz. Arch. Tierheilkd., 132, 289, 1990. 33. Halper, J., Grubman, M.J., and Jacobsen, K.L., Development of chicken antibodies to bovine interferon alpha, Immunol. Invest., 28, 19, 1999. 34. Hofmann, A. et al., Lipopeptides as adjuvants for the immunisation of laying hens, Altex, 13, 26, 1996. 35. Schwarzkopf, C. and Thiele, B., Effectivity of alternative adjuvants in comparison to Freund’s complete adjuvant, Altex, 13, 22, 1996. 36. Erhard, M.H. et al., Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens, Poult. Sci., 79, 1264, 2000. 37. Olbrich, C. et al., Stable biocompatible adjuvants — a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity, compatibility and efficacy in chicken, Altern. Lab. Anim., 30, 443, 2002. 38. Hedlund, G.P. and Hau, J., Oral immunisation of chickens using cholera toxin B subunit and Softigen as adjuvants results in high antibody titre in the egg yolk, In Vivo, 15, 381, 2001. 39. Kunz, U.S., Pospischil, A., and Paccaud, M.F., Immunohistochemical detection of chlamydiae in formalin-fixed tissue sections: comparison of a monoclonal antibody with yolk derived antibodies (IgY), Zentralbl. Veterinarmed. B., 38, 292, 1991. 40. Tini, M. et al., Generation and application of chicken egg-yolk antibodies, Comp. Biochem. Physiol. A Mol. Integr. Physiol., 131, 569, 2002. 41. Hacker, G.W., unpublished data 2, 2003. 42. Ferber, P.C. et al., The generation of monoclonal antibodies in mice: influence of adjuvants on the immune response, fusion efficiency and distress, Lab. Anim., 33, 334, 1999. 43. Lindl, T., Mass production of monoclonal antibodies: technical, scientific and ethical considerations, Altex, 5, 16, 1988. 44. Fischer, R.W. and Ferber, P.C., Monoclonal antibodies: comparative methods for in vitro production, Altex, 9, 15, 1992. 45. Falkenberg, F.W., Monoclonal antibody production: problems and solutions, Res. Immunol., 149, 542, 1998. 46. Hendriksen, C.F. and de Leeuw, W., Production of monoclonal antibodies by the ascites method in laboratory animals, Res. Immunol., 149, 535, 1998.
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47. Hendriksen, C.F. and de Leeuw, W.A., In vivo and in vitro production of monoclonal antibodies: current possibilities and future perspectives. Discussion, Res. Immunol., 149, 611, 1998. 48. Jackson, L.R. et al., Monoclonal antibody production in murine ascites. I. Clinical and pathologic features, Lab. Anim. Sci., 49, 70, 1999. 49. Jackson, L.R. et al., Monoclonal antibody production in murine ascites. II. Production characteristics, Lab. Anim. Sci., 49, 81, 1999. 50. Altweb, http://altweb.jhsph.edu/topics/mabs. 51. Kuhlmann, I.I., Kurth, W., and Ruhdel, I.I., Monoclonal antibodies: in vivo- and in vitro-production in laboratory scale with consideration of the legal aspects of animal protection, Altex, 6, 12, 1989. 52. Balls, M. and Corcelle, G., Statement on the scientific acceptability and practical availability of in vitro methods for the production of monoclonal antibodies, 1998, http://ecvam.jrc.it/publication/ MAb_statement.pdf. 53. National Institutes of Health, Production of monoclonal antibodies using mouse ascites method, in Office of Laboratory Animal Welfare Report 98-01, 1997, http://www.nal.usda.gov/ awic/newsletters/v8n3/v8n3oprr.htm. 54. U.S. National Research Council, Monoclonal Antibody Production, http://grants.nih.gov/ grants/policy/antibodies.pd, 1999. 55. National Health and Medical Research Council of Australia, Guidelines on Monoclonal Antibody Production, endorsed March 15, 2001, http:// www.ausinfo.gov.au/general/gen_hottobuy.htm. 56. Monash University of Australia, Guidelines and Policies: Monoclonal Antibody Production, 2001, http://www.monash.edu.au/resgrant/animalethics/guidpol/mabprod.html. 57. Lipman, N.S. and Jackson, L.R., Hollow fibre bioreactors: an alternative to murine ascites for small scale (10% of invasive tumor cells.
1+ 2+ 3+
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Molecular Morphology in Human Tissues: Techniques and Applications estimate of the intensity and percentage of cells stained (ICC) or the number of signals present (in situ-based techniques). When the accuracy of manual evaluation of HER2 results was assessed in a study in 2001, the level of agreement between the expert assessors and the evaluations from 67 other laboratories was found to be relatively poor (kappa 0.64, 95% CI, 0.57–0.71) [26]. However, the reproducibility of scoring was found to be significantly greater between those laboratories using a highly standardized assay system. Similarly, relatively poor agreement is generally seen in the manual scoring of breast carcinomas stained for hormonal receptors, particularly in the evaluation of tumors with relatively low amounts of ER or PR expression or when the pattern of hormonal expression is very heterogeneous. 18.6 HOW TO ENSURE REPRODUCIBLE, STANDARDIZED, AND FULLY QUANTIFIABLE IMMUNOCYTOCHEMICAL AND IN SITU-BASED ASSAYS Of the main factors considered preventing the implementation of reliable and fully quantifiable ICC and in situ-based assay, the vast majority can be addressed by the employment of stringent quality assurance (QA) measures. 18.7.1 Quality Assurance Quality assurance (QA) encompasses all measures taken to ensure the reliability of investigations, starting from satisfactory test sample selection and analyzing it appropriately, to recording the result accurately and reporting it to the clinician for appropriate action, with all procedures being documented for reference [27]. Two of the main features of QA are Internal 280
Quality Control (OQC) and External Quality Assessment (EQA). 18.7.1.1
Internal Quality Control
Internal quality control (IQC) is defined as the set of procedures undertaken by the staff of a laboratory for the continual evaluation of the reliability of the work of the laboratory and its emergent results, in order to decide whether they are reliable enough to be released on a day-to-day basis [27–29]. Most IQC procedures employ analysis of a control material and compare the result with predetermined limits of acceptability [27]. As with any laboratory assay, ideally all aspects of the ICC technique and the preparation of the tissues on which the assay is performed should be monitored by quality control procedures. 18.7.1.1.1 Internal Quality Control of Fixation and Tissue Preparatory Methods
There is currently no “standard” fixative; and while formaldehyde-based fixatives predominate in the U.K., formulations differ between laboratories and include 10% NBF, 10% formalin in tap water, 10% formal saline, and 10% NBF with saline [23, 24]. In addition, 10% formal acetic, Bouin’s fixative, Carson’s fixative, B5, and Dubosq Brazil are used in some laboratories offering an ICC service. Fixation time is difficult to standardize due to the different sizes of specimens entering the laboratory, as the penetration of fixative into large specimens will take longer than with small specimens. Tissue fixation has a significant influence on ICC as most antigens are altered during the process [24]. Prior to the advent of HIER, laboratories usually attempted to minimize the length of fixation, as prolonged fixation resulted in irretrievable loss of many antigens, particularly membrane-associated antigens such as CD20 and immunoglobulin (Ig) light
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Quality Assurance of Immunocytochemistry and Molecular Morphology chains [30]. However, lack of adequate fixation or delay in fixation may be equally detrimental to labile antigens. With respect to paraffin processing of tissues for ICC, in a survey of the schedules used by laboratories in the U.K., Williams et al. [31] found nearly as many different schedules as the number of laboratories participating in the survey. They subsequently investigated the effect of tissue preparation on immunostaining to establish whether a specific preparation schedule would allow for the optimal demonstration of all antigens. Of the fixatives tested, 10% formal saline, 10% NBF (except CD45RO), and 10% zinc formalin (except for CD3) gave the most consistent results overall and showed excellent antigen preservation. Of the fixatives previously recommended for ICC, 10% formal acetic, B5, and Bouin’s fixative all showed poor antigen preservation for the markers tested. The period of fixation and the pH of formalin also significantly affected the immunoreactivity of some of the antigens. However, the use of HIER was found to reduce or eliminate most of the weak staining observed in the study. Of the nine tissue processing factors investigated, only two had any significant effect on immunoreactivity. Increasing the temperature of processing from ambient to 45°C and longer processing times for dehydration and wax infiltration were both found to improve immunostaining. Type of processor, type and quality of reagents, time in clearing agent, use of vacuum, all of which had been suggested as possible causes of poor processing, were found to have no effect on the subsequent ICC. Williams et al. concluded that their findings, some of which were contrary to those of other studies, were explained by the variable response of different antigens to the effects of tissue preparation, and, hence, there was no standard universal tis-
sue preparation schedule for the optimal demonstration of all antigens [24]. The temperature and duration of section drying were found to affect the immunoreactivity for several antibodies, with drying at temperatures of greater than 60°C for greater than 4 hr being deleterious to the antigens investigated [24]. Consequently, it was recommended that, regardless of the antigen to be demonstrated, sections should be dried overnight at 37°C. If the sections are required more rapidly, or if greater adhesion is required prior to HIER, then drying at 60°C for up to 4 hr is recommended [24]. Some studies have found deterioration of antigens in stored sections [32–34] However, others have found no deterioration of the markers investigated, including estrogen receptors, CD3, CD20, CD45RO, vimentin, and Ig light chains in sections stored for up to 4 months at room temperature [24, 35]. 18.7.1.1.2 Internal Quality Control of Immunocytochemical Reagents
The primary antibodies, secondary detection systems, and reagents employed in the ICC assay are subject to in-house quality control by antibody manufacturers. Some have the International Organization for Standardization (ISO) 9001 standard [36]. The objective of the IS0 9000 series of standards is to give purchasers assurance that the quality of products provided by a supplier have met certain defined requirements. The standards set out a definitive list of features and requirements that should be a component of the organization’s management system and maintained through a system of procedures using documented policies and manual. These ensure that a defined quality is built into the process and achieved, regardless of the product manufactured, service provided, or the technology used [37]. 281
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Molecular Morphology in Human Tissues: Techniques and Applications The quality control of production, marketing, and use of antibodies for use in a clinical setting is currently influenced greatly by the United States (U.S.) Food and Drug Administration (FDA). Ruling on the classification and reclassification of immunochemical reagents and kits took effect on November 23, 1999 [38, 39]. From this date onward, the FDA expects U.S. laboratories not to use antibodies labeled “for research purposes only” in diagnostic tests, and that the results of studies using these reagents will not be accepted for reporting in patients’ clinical records [39]. While this is an U.S. ruling, most of the major antibody suppliers and producers in Europe have distribution networks and customers in the U.S. Consequently, antibodies and the respective kits produced by these companies, irrespective of their destination, are likely to conform to U.S. FDA requirements. In addition, since 2003, European legislation requires European producers of antibodies and ancillary reagents to conform to Directive 98/79/EC [40]. While this is a self-certification process for most markers, it requires all companies to have in place, from this date onward, quality assurance methods similar to that required for IS0 9000. A few products, however, such as the DakoCytomation HercepTest (a semi-quantitative ICC assay to determine HER2/neu protein overexpression in breast cancer tissues), require certification from an external body.
A small number of ICC tests are classified as Class II devices, of which assays for ER and PR are quoted as examples. These are markers of potential predictive or prognostic value and, as such, would also include markers such as p53 and HER2/neu [39]. For Class II devices, the manufacturer is required to submit data validating performance claims that may be derived from sponsor-supported studies or from existing literature. Similarly, Class III devices, which include ICC assays not obviously part of the surgical pathology diagnostic process and which may result in generation of a separate report to the clinician, require pre-market notification [38].
Under the new FDA ruling, the majority of antibodies used to assist in routine histopathological diagnosis are classified as Class I devices. ICC tests employing these antibodies are now recognized by the FDA as “special stains” that are “adjuncts to conventional histopathological diagnostic examination,” with results that are “incorporated into the diagnostic interpretation by the pathologist” and “are not usually
The quality assurance conducted by some leading antibody manufacturers in the production of Class II or III devices is more stringent than that for Class I produced by the same companies. For example, for the estrogen receptor clones 6F11, having passed through the basic stages of antibody production, undergoes further assays to ensure minimum batch-to-batch variation. An ELISA is used to measure the
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reported to the clinician” [38]. In the United States, it is expected that these tests are performed in high complexity laboratories certified under Clinical Laboratory Improvement Amendments (CLIA) of 1988 guidelines [41], to provide an additional level of insurance against undetected “device failure.” If an antibody or ICC reagent qualifies as a Class I device, then it is exempt from pre-market notification by the manufacturer and in so doing puts greater responsibility or accountability on the “user” to evaluate and document the efficacy of the antibody as an adjunct to diagnosis. Pre-market notification, requires the antibody producer to provide extensive documentation giving scientific evidence that supports the intended use of the device (assay).
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Quality Assurance of Immunocytochemistry and Molecular Morphology concentration of immunoglobulin in the bulk-diluted material against an approved sales batch. If the concentration is too strong or too weak, the strength is adjusted to the sales batch strength and another ELISA is performed to verify the concentration. When the concentration is correct, it is evaluated by ICC for a second time over a range of dilutions and matched against the previously QA approved sales batch. In the production of estrogen receptor clone 1D5, all new batches having had their immunoglobulin (Ig) concentration determined by single radial immunodiffusion, are tested on three to five mammary carcinomas (to include at least two lowexpressing estrogen receptor positive tumors) by ICC. The results are compared against a “gold standard” (an approved sales batch), both with respect to their Ig concentration and their ICC performance. In this way, batch-to-batch variation for these clones is minimized. Obviously, minimum batch-to-batch variations are essential for antibodies that are to be used in a semiquantitative ICC assay and particularly when marketed in a prediluted, ready-touse form. Without this stringent quality control, the ICC assay would be inaccurate, inconsistent, and with little scope for corrective adjustment by the purchasing laboratory. Similarly, with these assays it is crucial that there be minimal batch-tobatch variation in the sensitivity of the detection systems. 18.7.1.1.3 Internal Quality Control of the Exposure of Antigenic Epitopes and Nucleic Acids in Formalin-Fixed Tissues
The introduction of heat-induced epitope retrieval (HIER) in the early 1990s [42–44] greatly extended the number of markers that could be employed on routinely formalin-fixed and paraffinprocessed tissues and cells. This method effectively “unmasked” antigens previously
thought to be irretrievably lost in routinely fixed and processed tissue sections. This has allowed for their subsequent ICC demonstration by existing antibodies, previously reserved only for use on frozen sections. In addition, this technique appears to enhance the staining of antigens whose demonstration may have been badly hampered by excessive formalin fixation and for which any attempt to retrieve them by proteolytic enzyme digestion may have met with only limited success, or as in the case of some antigens, would have resulted in the destruction of the epitope (e.g., the epitope recognized by the B-cell marker, CD20) [45]. The initial experiments utilizing heat to expose antigens in formalin-fixed and paraffin-processed tissue sections were performed by Shi et al. in 1991 [42]. These experiments utilized the findings of early biochemical studies by Fraenkal-Conrat et al., who showed that the cross-linkages induced between formaldehyde and proteins could be reversed by heating at high temperature or by treating with strong alkali [46, 47]. Shi et al. [42], in applying these techniques to tissue sections, succeeded in obtaining increased ICC sensitivity after microwaving the sections in metal solutions. Other studies subsequently introduced the use of different heating instruments with apparently equal success and, in addition to microwave ovens, include pressure cookers [48], autoclaves [43], steamers [49], plastic pressure cookers inside microwaves [49, 50], and water baths and hot ovens [51, 52]. Some authors have advocated the use of low-temperature antigen retrieval, that is, overnight at 70 to 80°C. These authors investigated the use of this method with various antibodies, including ones to estrogen receptor, progesterone receptor, and HER2. They claimed that the results achieved were comparable to those obtained using high283
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Molecular Morphology in Human Tissues: Techniques and Applications temperature antigen retrieval and had the advantages of being safer and simpler [53, 54]. Experience to date shows that this method of antigen retrieval is most useful for markers for which excessive HIER may damage antigenic epitopes (e.g., those recognized by some HER2/neu markers). Most of the more popular devices used to effect HIER (e.g., pressure cookers and microwaves) are designed for domestic or catering use rather than for use in the laboratory. In this respect, it is unlikely that their warranty covers their scientific reliability. The method chosen is frequently dictated by factors such as the equipment locally available, the number of slides to be stained (the pressure cooker and autoclaves have larger slide capacities), and health and safety (e.g., domestic pressure cookers are considered a safety hazard) [55]. Some authors consider that the equipment used per se is not important and that the critical factor is the relationship between the maximum temperature achieved and the maintenance of sections at this temperature for an adequate period of time [10, 25, 56]. The study by Shi et al. [56] showed that the heating time required to achieve optimal results negatively correlated with the heating temperature, over the temperature range 60 to 100°C, when immunostaining with the proliferation marker MIB1 and a citrate buffer antigen retrieval solution. In this experiment, the duration of antigen retrieval for the optimal result ranged from 20 min at a temperature of 100°C to 10 hr at 70°C [56]. Dry heat alone is not effective in the antigen retrieval process and an appropriate aqueous solution must be employed. On the whole, heavy metal solutions as used in the initial experiments by Shi et al. [42] or similar potentially toxic chemicals 284
are not widely used because of the health and safety implications. For the majority of antigens, sodium citrate, Tris-HCl, phosphate or sodium acetate buffers are reported as being effective and that staining intensity differs little over a pH range of 1 to 10 [55, 57]. While Shi et al. [56] have claimed to achieve good results with MIB1 using this buffer, some authors investigating the same antigen have failed to achieve retrieval when using tissues subjected to a slightly different fixation and paraffin processing regime [58]. In the study by Pileri et al. [59], for many of the antigens investigated, Tris-HCl was found superior to citrate buffer; however, both were considered less efficient than EDTA (etheylenediaminetetraacetic acid) buffer, particularly with the antibodies to nuclear antigens tested such as MIB1, estrogen receptors, and progesterone receptors [59]. The rationale behind using a known calcium chelating agent such as EDTA is based on the work of Morgan et al. [60], who postulated that tight complexing of calcium ions or other divalent metal cations with proteins during formalin fixation is responsible for masking certain antigens. The chelating properties of the buffers employed are consequently thought to aid in their removal during the HMAR process, with high temperature providing sufficient energy to release the calcium ions from the multivalent cage-like complexes that they form with tissue proteins [60]. A few antigens are reported to show significant differences in retrieval patterns with differing pH values. MIB1 and estrogen receptors are reported to show a Vshaped response, with greater intensity of staining at the extreme ends of the pH range. Others, including HMB45 and MT1, show greater intensity with increasing pH [57]. These differences are not understood but may be related to the
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Quality Assurance of Immunocytochemistry and Molecular Morphology amino acid sequence of the epitope [55]. To find the optimal pH for new antibodies, Shi et al. have suggested the use of a “test battery” approach, similar to a “checkerboard” titration used for determining optimal antibody dilutions [56, 57]. The new antibody is incubated with slides that have undergone pretreatment with a number of different combinations of temperature and pH. This allows selection of the optimal conditions on the basis of the slide showing optimal staining. The authors suggest that if satisfactory results are not achieved with the standard citrate or Tris-HCl buffer, then less commonly used solutions should be tested. The marker and fixative employed dictate whether or not to employ antigen retrieval techniques. Some antigens benefit from, or necessitate, a pretreatment stage employing proteolytic enzyme digestion, HIER, or no pretreatment at all. Without guidance on which form of antigen retrieval is best for a given antigen, individual laboratories are left to themselves to decide which method appears most appropriate, although frequently the suppliers or producers of antibodies will give some indication as to which pretreatment step is appropriate. With respect to the demonstration of hormonal receptors in breast cancer, it has been shown that inefficient HIER is frequently the main factor responsible for poor-quality results in multiple cancer centers; and when this factor is addressed, significant improvement in results is seen and subsequently maintained [10, 25]. Although some authors recommend HIER as a standard procedure prior to the demonstration of most antigens [61], others recommend a more conservative approach [49, 62]. While there is no nationally or internationally recognized standard antigen retrieval protocol, internal
standardization of the technique is required if the results produced by a laboratory are to be reliable and reproducible. Variables that require standardization in the antigen retrieval technique include the choice of heating method (e.g., pressure cooker, microwave, etc.), retrieval solution, pH, temperature, and duration of heating. The test battery approach suggested by Shi et al. [56] can be used to also establish not only the optimal buffer pH and temperature of heating, but also the optimal retrieval solution and duration of heating, for a given antigen, by holding the variables of pH and temperature of heating constant. With respect to enzymatic proteolytic antigen retrieval, the choice of enzyme usually dictates the temperature and pH of the solution, as different enzymes have different preferential pH and temperature values. For example, the optimal values for a mammalian-derived trypsin are set to be pH 7.8 at 37°C, with 0.1% calcium chloride included as an activator [63]. Proteolytic enzymes (e.g., proteinase K) for in situ techniques are used not to expose antigenic epitopes, but to reverse some of the cross-linking of proteins to allow entry of nucleic acid probes so that they can hybridize with the complementary sequence for the biomolecule of interest. 18.7.1.1.4 Improvement in the Reproducibility of Evaluation of Assay Results
While manual evaluation of ICC and in situ-based results have generally shown poor interobserver agreement and reproducibility, the use of automated image analysis has the potential to reduce this variability. In a study comparing the results of manual evaluation to that achieved with automated image analysis on human ovarian and breast carcinoma cell lines, image analysis was highly reproducible compared to manual scoring and generated readings 285
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Molecular Morphology in Human Tissues: Techniques and Applications that were significantly different for each range of values for HER2 stained cell lines categorized by experienced assessors as 3+, 2+, 1+, and 0 (Figure 18.1) [64]. The range of linear readings for each category with automated image analysis also gave different and quantifiable readings for “weak” and “strong” results within each category. Future work will look at whether these differences (and readings that lie between the 3+ and 2+ categories) are relevant to clinical cases, with respect to correlation with gene amplification and relative response rates, when employing a highly standardized assay. In contrast, manual scoring, wherein results are merely placed in one of several categories (Tables 18.1 and 18.2), appears somewhat primitive when compared to the level of sophistication possible with automated image analysis to generate specific and repro-
ducible sets of linear readings (Figure 18.1). Using a reliable automated image analyzer, more accurate cut points can be established for markers such as HER2 and ER and PR. For example, the 10% value used as one of the parameters in the current manual scoring system for HER2/neu does not have any bearing on likely clinical response to Herceptin but was included merely to accommodate the vagaries of formalin fixation. Because automated image analysis can provide fully quantitative metric readings on each case, it has the potential to provide more accurate cut-points to determine those patients most likely to respond to Herceptin therapy. Similarly, with respect to prognosis, a recent, large study of patients with node-negative breast cancer showed HER2/neu expression as a significant discriminant of prognosis in a subgroup of patients with rapidly prolifer-
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No. of stained fragments (95% CI)
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Figure 18.1 Image analysis readings for the number of immunostained membrane fragments for HER2/neu, for the ovarian and breast carcinoma cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 manually scored as 3+, 2+, 1+, and “0.” Error bars show mean values with 95% confidence intervals.
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Quality Assurance of Immunocytochemistry and Molecular Morphology ating tumors, with significantly higher relapse when tumors were highly positive for HER2/neu (≥30%) [65]. The ability to more accurately and readily record numerous objective parameters such as the extent and intensity of membrane staining by automated systems will surely be valuable for both prognosis and prediction in the future. 18.7.1.1.5
Standard Reference Materials
Adequate quality control of predictive markers in cellular pathology that require quantitation is hampered by the lack of known and approved “standards” by which assays can be gauged to guard against oversensitivity (false-positive results), and low sensitivity (false-negative results). The use of tumors for this quality control is not ideal, as the amount of expression will always vary between one tumor and anoth-
er; consequently, unless all laboratories use the same tumor to standardize their assays, no two laboratories are likely to produce precisely the same assay sensitivity. The use of the same tumor by all institutions is obviously impossible due to the limited availability of tumor material. In addition, obtaining any tumor material for QC is aggravated by recent adverse media publicity on the issue of organ retention [66, 67]. One solution to the problem that has been proposed by various sources is to use cell line controls that have been formalin fixed and paraffin processed and therefore preserved in the same way as routine pathology tissues [18–20, 26, 68]. Cell lines have the advantage of being available in abundant quantity and if a large culture is harvested, it will provide abundant quality control material with a standard level of protein expression (Figure 18.2).
Figure 18.2 Formalin-fixed and paraffin-processed sections of cell lines immunostained for HER2/neu: (A), 3+ staining of the ovarian carcinoma cell line SKOV-3; (B), 2+ staining of the breast carcinoma cell line MDA-MB-453; (C), 1+ staining of the breast carcinoma cell line BT-20, (D) “0” staining of the breast carcinoma cell line MCF-7 [19].
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Molecular Morphology in Human Tissues: Techniques and Applications To highlight the need for such standards, in 2002 a workshop was organized by the U.S. National Institute of Science and Technology (NIST), the Food and Drug Administration (FDA), the National Cancer Institute (NCI), and the College of American Pathologists (CAP) aimed at identifying a suitable and nationally agreed-upon reference material (standard) for the HER2/neu assay, in the hope that this would assist in providing the muchneeded level of standardization required for this test [68]. It was agreed that such a standard was desirable and necessary to ensure the reliability of HER2 testing to qualify patients for Trastuzumab therapy. Two standards consisting of well-characterized cell lines will be produced: one will be a NIST-certifiable standard and the other will be a commercially developed standard for use in all HER2 testing. The aim is that this approach will act as a template for future, potentially valuable predictive markers. 18.7.1.2
External Quality Assessment
The principal objective of External Quality Assessment (EQA) is to detect differences in assay results between laboratories and provide guidance on how to achieve the standards deemed more universally “acceptable.” EQA is defined by the World Health Organization and the European Committee for Clinical Laboratory Standards as a system of retrospectively and objectively comparing results from different laboratories by means of an external agency [28, 29]. While provision of standard reference materials (SRMs) for markers such as HER2 is highly desirable, one must remember that this is only material upon which it is possible to achieve a “standard result.” To actually produce the “standard result,” a total comprehensive QA package 288
is required that provides laboratories with the necessary guidance and support on how to achieve appropriate results with the assays and how to best interpret and score them. This will assist in the reproducibility of the results of clinical trials and ensure that they are reliable and accurate, and is essential if appropriate threshold values for the assays are to be established from pooled clinical data emanating from multiple centers. This need is evident not only for groups involved in translational research and clinical trials, but also for clinical laboratories when the assays start to be routinely used. The controversy surrounding the lack of reproducibility of HER2 ICC assays was no doubt partly responsible for the delay in transition of the drug Herceptin from clinical trial stage to approved therapy by the United Kingdom National Institute for Clinical Excellence (NICE) [70]. In a similar way, a few years ago there was great excitement about the potential clinical importance of p53 alterations. Multiple assays were used to test for p53, including ICC employing several different antibodies and antigen retrieval methods, PCR/single-strand conformation polymorphism (SSCP), and direct sequencing for p53 mutational status. Subsequently, there was lack of uniformity of the cut-point, the method of reporting results, and the criteria for determining a positive result. Consequently, despite a plethora of reports about p53 alterations in various tumors, little in the way of clear evidence of its value as a tumor marker has emerged for any tumor site. The lack of a standardized assay to measure p53 status has contributed to much of this confusion [69]. If assay reproducibility cannot be assured, then it is likely that promising new therapies that rely on these assays will fall by the wayside and not progress from clinical trial status to approved drug status. The way to
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Quality Assurance of Immunocytochemistry and Molecular Morphology ensure a high level of assay reproducibility for new predictive markers is to instigate a comprehensive approach to QA, one that advocates the use of standard reference materials, highly standardized assay procedures, and participation in national EQA programs to provide guidance to laboratories achieving inappropriate results. 18.8 THE FUTURE Attention to stringent QC procedures, the use of standard reference materials, automated procedures for performing the assays, and their evaluation and participation in EQA programs will provide much of the framework to make standardization and accurate quantitation of ICC and in situ-based predictive assays possible. However, unless it is possible to gain a fuller understanding of the processes involved in formalin fixation, quantitation of any assay on formalin-fixed material will remain, to a certain extent, imprecise. Without full knowledge of the nature of fixation, it will not be possible to accurately determine how much fixation has taken place, and therefore how many of the biomolecules of interest have been preserved and precisely how much antigen retrieval is required to optimally expose the antigens or nucleic acids in the tissue of interest. Work on unraveling the precise nature of formalin fixation and the production of model systems that allow for determining the contribution of each step in the ICC assay to the final signal is part of an ongoing project utilizing synthetic peptides with known amino acid structure. Inclusion of the peptides with a specimen during the preparatory stages of formalin fixation and paraffin processing could also be used as a QC system to check on day-today variations in staining that are due to
inconsistencies in the preparatory steps of fixation and processing [71]. Further research and development will be required in the development of sophisticated image analysis systems that can accurately measure not only the intensity of staining, but also its distribution (e.g., membrane, nuclear, or cytoplasmic) and no doubt these automated systems will become commonplace in the evaluation of ICC and in situ-based assay results. More accurate and sophisticated detection systems are also likely to develop; among these, the use of semiconductor quantum dots (QDs) are among promising emerging fluorescent labels for cellular imaging. QD 560 and QA 608 have been linked to streptavidin as a label to replace peroxidase and alkaline phosphatase and used to immunostain for HER2 in both cell lines and tissue sections [72, 73]. The signals were brighter and considerably more photostable than comparable organic dyes; and using QDs with different emission spectra, it was possible to detect two cellular targets with one excitation wavelength. Such systems may provide for greater accuracy for in situ techniques using fluorescent labels such as FISH. Finally, there will be a growing need for QA at all stages of assay development — from the initial research investigation of a potential predictive marker through to its development in clinical trials and eventual use as a nationally approved assay. Only then can it be ensured that valuable markers do not fall by the wayside because of spurious results caused by diverse and inappropriate methodologies, that pooled data from clinical trials involving multiple centers is reliable, and ultimately that patients receive the most appropriate therapy irrespective of where they are treated. 289
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58. Morgan, J.M., Navabi, H., and Jasani, B., Role of calcium chelation in high temperature antigen retrieval at different pH values, J. Pathol., 182, 233, 1997. 59. Pileri, S.A. et al., Antigen retrieval techniques in immunocytochemistry: comparison of different methods, J. Pathol., 183, 116, 1997. 60. Morgan, J.M. et al., Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval, J. Pathol., 174, 301, 1994. 61. Leong, A.S.-Y., Cooper, K., and Leong, F.J.W.M., Appendix 2: Heat induced epitope retrieval and antigen retrieval protocol, in Manual of Diagnostic Antibodies for Immunohistology, Leong, A.S.-Y., Cooper, K., and Leong F.J.W.-M., Eds., Greenwich Medical Media Ltd., London, 1999. 62. Swanson, P.E., Editorial: HIERanarchy: the state of the art in immunocytochemistry, Am. J. Clin. Pathol., 107, 139, 1997. 63. Huang, S.-N., Minassian, H., and More J.D., Application of immunofluorescent staining on paraffin sections improved by trypsin digestion, Lab. Invest., 35, 383, 1976. 64. Rhodes, A. et al., The use of cell line standards and external quality assessment to standardise assays for HER2/neu in European cancer centres and the potential of automated image analysis to provide more accurate cut-points for predicting clinical response to trastuzumab, Breast Cancer Res. Tr., 82, S76, 2003. 65. Volpi, A. et al., HER-2 expression and cell proliferation: prognostic markers in patients with nodenegative breast cancer, J. Clin. Oncol., 21, 2708, 2003. 66. Hunter, M., Alder Hey report condemns doctors, management, and coroner, BMJ, 322, 255, 2001. 67. Bauchner, H. and Vinci, R., What have we learnt from the Alder Hey affair? BMJ, 322, 309, 2001. 68. Hammond, M.E.H. et al., Standard reference material for Her2 testing. Report of a National Institute of Standards and Technology-sponsored Consensus Workshop, Appl. Immunohistochem. Molec. Morphol., 11, 103, 2003. 69. Hammond, M.E.H. and Taube, S.E., Issues and barriers to development of clinically useful tumour markers: a development pathway proposal, Semin. Oncol., 29, 213, 2002. 70. National Institute for Clinical Excellence (NICE), 11 Strand, London WC2N 5HR, U.K. Web: www.nice.org.uk
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Index A Adjuvants, 254–256, 258 AEC (aminoethyl carbazol), 4, 45–46, 56–57, 59 Aldehyde-containing reagents, quenching, 87–88, 90 Alexa dyes, 15, 16, 17, 226–227 Alexa Fluor labels, 14, 85, 95–96 Alkaline phosphatase, 66 commercial visualization systems, 59 double immunostaining systems, 8, 9 endogenous activity, 5, 48, 78 fluorescent end-products, 66–67 fluorogenic substrates, 17 multiple antigen immunohistochemistry, 4 multiple staining protocols, 58 order of multiple enzymatic development, 49 Alkaline phosphatase anti-alkaline phosphatase (APAAP), 55 AMCA, 14–15, 17, 19, 218 Aminoethyl carbazol (AEC), 4, 45–46, 56–57, 59 7-Amino-4-methylcoumarin-3-acetic acid (AMCA), 14–15, 17, 19, 218 Amphoterin, 92 Amplex Red, 18 Animal models, whole-mount ISH, 157–161 Animal Research Kit (ARK), 38–39, 43, 50, 56 Animal welfare, reagent production issues, 253–270 adjuvants, 254–256, 258 alternative antibody production methods, 260–263 ascites method, 258–260, 268, 269 dignity of animals, 269 fetal calf serum, 264 legal aspects, 264–269 monoclonal antibodies, 258–260 pain medication, 255 polyclonal antibodies, 254–258 Antibody conjugate selection, 41–43 Antibody cross-reactivity, See Cross-reactivity Antibody elution, 2–6, 32 Antibody production, animal welfare considerations, 253 adjuvants, 254–256, 258 ascites method, 258–260, 268, 269 chicken eggs, 256–258 European regulations, 268–269 molecular morphology, 258 monoclonal antibodies, 258–260 plants, 263 polyclonal antibodies, 254–258 quality control, 282 recombinant technology, 260–264 Antigen co-localization immunostaining, See Multiple immunostaining techniques
Antigen retrieval fixation and, 284–285 heat-induced epitope retrieval (HIER) systems, 278, 281, 283–285 Southwestern histochemistry, 143 Anti-sense cRNA probe, 158, 161–162 Anti-viral vaccination, 179 Apoptotic marker visualization, 199–208 biopsy vs. postmortem tissue, 200 BrdU immunohistochemistry, 203–204 immunostaining protocols, 202–205 immunostaining results and discussion, 205–208 in situ end labeling, 199, 207 staining procedure, 202–206 tissue embedding in Epon, 201–202 tissue fixation and dehydration, 200–201 Applied Immunohistochemistry and Molecular Morphology, 107 Argentaffin-type reactions, 116 Array-based comparative genomic hybridization, 123–132, See also Comparative genomic hybridization Ascites method, 258–260, 268, 269 Austrian animal welfare laws, 268 Automated image analysis, 285–286 Automated immunohistochemical staining systems, 8 Automated mRNA in situ hybridization, 147–155 controls, 154 hybridization temperature, 154 instrumentation, 142–143 probe design, 154 riboprobe system, 150–151 sample screening in microarrays, 152–153 signal amplification, 155 specificity, 154 tissue fixation, 151–152 tissue qualification, 154 whole-mount ISH protocol, 163–166 Autometallography, 81, 115–117, See also Gold cluster labels; Immunogold-silver staining; Nanogold; specific types autonucleation, 84 enzyme metallography, 82, 86–87 gold enhancement, 84, 89–91, 118 light sensitivity, 83 particle size, 83–84 pH and, 116 process, 83 quenching aldehyde-containing reagents, 87–88, 90 reaction quenching, 117 silver enhancement, 82–84, 87–89, 109, 117–118
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Molecular Morphology in Human Tissues: Techniques and Applications tyramide-amplified Nanogold-silver/gold ISH, 108–120 Autonucleation, 84 Autoradiography, 9 Avidin-biotin complex (ABC), 7, 12
B Background fluorescence, 16 Background staining, CARD and, 7 Bacterial artificial chromosome (BAC) arrays, 124–125 Benchmark system, 149 Biopsy tissue, apoptosis indicators, 199–208, See also Apoptotic marker visualization BrdU immunohistochemistry, 203–204 embedding in Epon, 201–202 fixation and dehydration, 200–201 Biotin, endogenous activity, 7, 48 Biotinylated tyramides, 55, 110 Biotinylation, 7, 41 Blue BB, 45–46 Blue fluorophores, 14–15 Bluo-Gal, 59 BODIPY, 14, 16 BrdU immunohistochemistry, 203–204 Breast cancer HER2 gene amplification detection, 94, 101, 113, 123–124 HER2/neu expression and prognosis, 276, 286–287 Brown-red color combination, 45 Brown tissue pigments, 49
C Cancer, breast, See Breast cancer Cancer, genomic analysis of, 123–132, See also Comparative genomic hybridization Carbocyanine (Cy 2), 14, 19 Carboxyrhodol (Rhodol Green), 14 CARD, See Catalyzed reporter deposition Cascade Blue, 15 Catalyzed reporter deposition (CARD), 7, 108, See also Tyramide signal amplification background staining, 7 double-CARD procedures, 12, 21 double-immunofluorescence staining, applications, 10, 11–12 double immunofluorescence staining, protocol, 20–21 GOLDFISH, 102, 104 performance factors, 119 peroxidase substrates, 68 tyramide-amplified Nanogold-silver/gold ISH, 110–113 Catalyzed signal amplification (CSA), 7, 10, 108, See also Tyramide signal amplification CD117, 276 Cell culture
296
fetal calf serum supplements, 264 Southwestern histochemistry protocol, 137–138 Ceroid pigment, 49 Chicken eggs, 256–258 Chinese medicine, 255 4-Chloro-1-naphthol, 4 Chromogens, See also specific stains multiple immunoenzymatic staining system, 43 peroxidase substrates, 4 selection for multiple immunostaining, 5 Chromosome enumeration probe (CEP), 170 Citrate buffer, 117 Class I devices, 282 Class II devices, 282 Class III devices, 282 Clinical Laboratory Improvement Amendments (CLIA), 282 CODFISH, 102 Colloidal gold, 81–82, 217, See also Gold cluster labels double immunostaining system, 8–9 Colloidal silver, double immunostaining system, 9 Co-localization, double staining approaches, See Multiple immunostaining techniques Comparative genomic hybridization (CGH), 123–132 array systems, 125 bacterial artificial chromosome (BAC) arrays, 124–125 DNA isolation and quantitation, 125–126 DNA labeling, 126–128 hybridization protocol, 128–132 microarray washing protocol, 130–131 troubleshooting, 132 visualization protocol, 131–132 Complete Freund’s adjuvant (CFA), 254 Computerized imaging, 46–47 Confocal laser scanning microscopy (CLSM), 14, 16, 91, 209, 230–231 Cot-1 DNA, 170 Coverslipping glues, 117 Cross-reactivity, double immunofluorescence techniques and, 9–12 Cryostat section protocol, 51–52 Cy 2, 14, 19 Cy 3 (indocarbocyanine), 14, 19, 67, 95 Cy 5 (incodicarbocyanine), 14, 19 Cytogenetics, 124
D DAB (3,3′-diaminobenzidine tetrahydrochloride), 2, 4, 18, 32–33 double immunoenzymatic staining protocol, 57 Southwestern histochemistry, 134 tissue pigmentation, 49 DABCO, 16 DakoCytomation Animal Research Kit, 38, 56 Dehydration, 201 Dextran-based polymeric systems, 7–8, 97
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Index 3,3′-Diaminobenzidine tetrahydrochloride, See DAB o-Dianisine, 4 1,4-Diazabicyclo[2.2.2]octane (DABCO), 16 Differential interference contrast (DIC), 210 Digital optical microscope (DOM), 210–221 commercial sources, 211, 217 fluorescent labels, 218, 219 general biological research, 219 immunohistochemistry applications, 217–218 materials and methods, 217 molecular morphology, 218–219 system, 211–212 3D-3D co-localization, 210–211, 219 Digoxigenin-labeled probe, 134, 158, 161–162 Dinitrophenyl (DNP), 119 Dipalmitoyl phosphatidyl ethanolamine (DPPT)Nanogold, 93 Direct/direct double staining approach, 34–35, 53 Direct immunofluorescence techniques, 9–10, 168 Discovery system, 149–150 DNA-binding proteins (transcription regulatory factors), 133–145, See also Southwestern histochemistry DNA denaturation, 183 DNA extraction, 180 DNA fragmentation, apoptotic indicators, 199, See also Apoptotic marker visualization immunostaining protocols, 202–205 immunostaining results and discussion, 205–208 post-mortem tissues and, 200 DNA isolation, 125 DNA microarrays comparative genomic hybridization, 123–132 gold cluster labels, 91–92 labeling, 126–128 DNA polymerase, 182, 184, See also Polymerase chain reaction Double-CARD, 12, 21 Double gold labeling, 86 Double immunofluorescence methods, 9–18, See also Immunofluorescence methods; Multiple immunostaining techniques protocols, 20–21 Double peroxidase-anti-peroxidase (PAP) staining, 6–7 Double-stranded oligo-DNA (ds-oligo DNA), 134 DsRedE5, 15 Dual parameter flow cytometry, 239–240 DuHuo, 255
E Eggs, hen, 256–258 Electroless deposition, See Autometallography Electron microscopy correlative fluorescence, 96–97 gold particle labeling applications, 82, 91 ELF-97, 17, 67, 71–75, 77–78 Elution techniques, 2–6, 32 cross-reactivity, 5
Endogenous enzyme activity, 4–5, 48–49, 78 Enhanced Polymer One-Step (EPOS), 6, 37, 55 EnVision doublestaining system, 34, 35 Enzyme-based fluorescence amplification, 65–80 advantages, 66 applications and results, 75 combined TSA and quantum-dot detection, 77 coupling reagents, 66–67 ELF-97, 17, 67 IHC protocols, 71–73 ISH protocols, 73–75 problems and technical issues, 77–78 multi-labeled TSA detection, 77 near-ultraviolet illumination, 67 sensitivity, 66 TSA detection methods, 76–77 TSA protocols, 69–71 Enzyme-labeled-fluorescence-97 (ELF-97), 17, 67, 71–75, 77–78 Enzyme-linked immunoadsorbent assays (ELISA), 66 Enzyme metallography, 82, 86–87 HER2 gene amplification assay, 102 Epidermal growth factor receptor (EGFR), 144, 276 Epon, 201–202 EPOS, 6, 37, 55 Estrogen receptor, 277, 282–283 Ethylene-vinyl acetate co-polymer, 256 European Union, animal welfare laws, 266–268 External quality assessment (EQA), 288–289
F Fab’ antibodies, 11, 20 Nanogold and fluorescent labeling, 95 platinum labeling, 97 Fast Blue, 43, 45–46, 58 Fast Red, 17, 45–46, 58, 59, 66 Federal Animal Welfare Act, 265 Fetal calf serum (FCS), 264 Fibroblast growth factor 8 (FGF 8) mRNA, 158 Film in situ zymography (FIZ), 245–251 analysis of proteolytic activity, 249 fixation, 246 immunogold-silver staining, 246–247 immunohistochemistry, 250 incubation protocol, 246 inhibition effects, 249–250 optimal incubation time, 248–249 staining protocol, 247–248 FISH, See Fluorescence in situ hybridization FITC, See Fluorescein isothiocyanate Fixation antigen retrieval and, 284–285 automated mRNA ISH, 151–152 biopsy tissue for apoptosis markers, 200–201 film in situ zymography, 246 internal quality control, 280–281 metallographic HER2 gene amplification assay, 103 protocol for in situ PCR, 191–193
297
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Molecular Morphology in Human Tissues: Techniques and Applications Southwestern histochemistry, 143 standardization issues, 277–278, 280, 289 whole-mount ISH, protocols, 158–161 Flow cytometry, signal transduction visualization, 239–240 Fluorescein isothiocyanate (FITC), 119 background fluorescence and, 16 digital optical microscope applications, 218 double-staining applications, 35, 37 multiple immunostaining applications, 14 peroxidase-based double immunostaining system, 8 quadruple immunofluorescence staining, 19 Fluorescence amplification, See Enzyme-based fluorescence amplification Fluorescence microscopy, See also Immunofluorescence methods combined gold labeling applications, 84–85, 94–97, 109 correlative electron microscopy, 96–97 double immunofluorescence methods, 9–18 filters, 17 Fluorescence in situ hybridization (FISH), 167–169 CODFISH, 102 direct and indirect, 168 HER2/neu amplification, 276 gold-facilitated, See GOLDFISH interphase cells, 168 metallographic HER2 gene amplification assay, 101–106 probe binding specificity, 169 probe types, 170 target accessibility, 169 thin-layer liquid-based preparations, 167–177 common problems, 176–177 hybridization protocol, 175 materials and equipment, 171–174 rapid wash procedure, 175–176 sample pretreatment, 174–175 3D-imaging using digital optical microscope, 211 Fluorescence quenching, 13, 16, 30 Fluorescence resonance energy transfer (FRET), 13, 85, 92–93 Fluorescent Activated Cell Sorter applications, 35 Fluorescent signal “bleaching,” 102 Fluorogenic enzyme substrates, 17–18 Fluoro-Nanogold, 84, 109 Fluorophores double immunofluorescence applications, 13–16 pH sensitivity, 15 time-dependent expression, 15 water-soluble (FM dyes), 15–16 FM dyes, 15–16 Forensic applications, 181–182 Formaldehyde/formalin fixation, 277–278, 280 antigen retrieval and, 284 DNA microarrays and, 125 standardization issues, 279 Formazans, 4
298
Förster distance, 85 Freund’s adjuvant, 254–255 Frozen tissue sections, Southwestern histochemistry protocol, 135–136
G β-Galactosidases, 4 commercial visualization systems, 59 double immunoenzymatic staining protocols, 58–59 fluorogenic substrates, 18 Gambusia affinis affinis, 97 Gelatinase expression, in situ zymography, 245–251 GenBank, 182 Genomic analysis of cancer, 123–132, See also Comparative genomic hybridization German animal welfare laws, 268 GH-1 probe, 138–140 GH3 culture cells, 133, 139–140 Glivec, 278 Glucocorticoid responsive element (GRE), 134, See also Southwestern histochemistry Glucose oxidase, 4, 5 Glucose oxidase-anti-glucose oxidase (GAG) system, 8 Glues, coverslipping, 117 Gold cluster labels, 81–100, See also Colloidal gold; Nanogold advantages, 81–82 autometallographic enhancement, See Autometallography combined fluorescent labeling applications, 84–85, 94–97, See also GOLDFISH correlative light and electron microscopy, 91 detection methods, 83 digital optical microscope applications, 217–218 double immunostaining system, 8–9 double labeling, 86 electron microscopy, 82 enzyme metallography, 86–87 IGSS, See Immunogold-silver staining in situ hybridization application, 93–94 in situ PCR application, 93 label characteristics, 82 lipid/liposome labeling, 92–93 microarray and biochip applications, 91–92 particle size distribution, 83–84 three-dimensional imaging applications, 210 GoldEnhance developer, 116 Gold enhancement, 84, See also Autometallography; Gold cluster labels ISH application, 94, See also GOLDFISH protocol, 89–91, 118 Gold-facilitated autometallographic in situ hybridization, See GOLDFISH GOLDFISH, 94 HER2 gene amplification assay, 94, 102, 104, 113 study set, 115 tyramide-amplified Nanogold-silver/gold ISH, 113–115
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Index Green fluorescence protein (GFP) conjugated systems, 219, 225, 226, 229, 231–233, 235, 239–242 Growth hormone (GH) transcription factors, 133, 134, 139–141, 143
H Heat antigen retrieval, Southwestern histochemistry, 143 Heat-induced epitope retrieval (HIER) systems, 278, 281, 283–285 Helicobacter pyroli, 144 HER2 gene amplification, 101–106, 123–124 antigen retrieval, 283–284 automated image analysis, 286 GOLDFISH, 94, 102–106, 113 cancer prognosis and, 276, 286–287 Class II device, 282 correlation with protein over-expression, 276 quantum dots, 289 standardization issues, 275, 288 Herceptin, 101, 276, 286, 288 High-throughput genetic analysis, 147–155, See also Automated mRNA in situ hybridization HIV-1, 179, 180 Horseradish peroxidase (HRP), 66 commercial visualization systems, 59 double immunoenzymatic staining protocols, 56–58 enzyme metallography, 86–87 fluorescent end-products, 66–67 multi-labeled TSA detection, 77 multiple antigen immunohistochemistry, 2, 4 order of multiple enzymatic development, 49 Southwestern histochemistry, 134 substrate dimerization, 68 tyramide signal amplification, 67 p-Hydroxyphenyl-containing peroxidase substrates, 67
I IgG biotinylation, 7 cross-reactivity in multiple immunostaining, 12 glass bead opsonization, 226–228 sheep red blood cell opsonization, 228–229 IGSS, See Immunogold-silver staining (IGSS) IgY, 257–258 Image analysis, automated, 285 Immunization, animal welfare issues, 254–256 Immunocytochemical reagents, internal quality control, 281–283 Immunocytochemistry, quality assurance of, See Quality assurance Immunoenzymatic methods, See also Immunostaining; Multiple immunostaining techniques adapting systems for other detectors or chromophores, 49 antibody elution techniques, 2–6, 32 multi-enzyme methods, 4–6
single enzyme methods, 2–4 antibody reagent production, See Antibody production, animal welfare considerations antibody tracers, 44–45 commercial visualization systems, 59 DakoCytomation Animal Research Kit, 38–39, 43, 50, 56 detection systems, 43–44 direct/direct system, 34–35, 53 double and multiple staining, 27–62, See also Multiple immunostaining techniques color combinations, 45–46 concept choice, 41–43 control, 40–41 general strategy, 39–40 indirect/direct format, 37–39, 54–56 indirect/indirect system, 35–37, 53–54 order of enzymatic development, 49 quadruple staining, 18 sequential techniques, 2, 32–34, 52–53 endogenous enzyme activity and, 4–5, 48–49, 78 fluorogenic substrates, 17 immunoreagent list, 56–57 non-commercial visualization systems, 56–69 AP/Fast Blue/Fast Red, 58 AP/New Fuchsin, 58 β-galactosidase/Bluo-Gal, 59 β-galactosidase/X-gal, 58–59 HRP/AEC, 56–57 HRP/DAB, 57 HRP/TMB, 57–58 requirements for successful performance, 30–32 standardization issues, 279 Immunofluorescence methods background fluorescence, 16 digital optical microscope applications, 218, 219 double and triple staining, 9–18, See also Multiple immunostaining techniques CARD-based, 10, 12, 21 cross-reactivity, 9–12 direct and indirect techniques, 9–10, 168 enzyme substrates, 17–18 Fab antibodies, 11, 20 fluorophores, 13–16 microscope filters, 17 paraformaldehyde vapor, 11, 21 protocols, 20–21 drawbacks, 30 FISH, See Fluorescence in situ hybridization; GOLDFISH fluorescence amplification, See Enzyme-based fluorescence amplification fluorophores, 13–16 PAP-combined triple staining, 18 quadruple immunofluorescence staining, 19 quenching, 13, 16, 30 time-dependent fluorophore expression, 15 triple immunofluorescence staining, 20
299
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Molecular Morphology in Human Tissues: Techniques and Applications water-soluble dyes, 15–16 Immunoglobulin G, See IgG Immunoglobulin purification, 41 Immunogold-silver staining (IGSS) digital optical microscope applications, 217–218 double staining applications, 9, 33 film in situ zymography, 246–247 three-dimensional imaging applications, 210 Immunohistochemistry (IHC) techniques, See also Immunostaining; specific applications, reagents, techniques automated staining systems, 8 consecutive sections, 1–2 digital optical microscope applications, 217–218 double and multiple immunostaining, See Multiple immunostaining techniques enzyme-labeled-fluorescence-97 protocols, 71–73, 78 enzyme metallography, See Enzyme metallography film in situ zymography, 250 fluorescence amplification for, 65–80, See also Enzyme-based fluorescence amplification general protocols, 51–52 cryostat sections, 51–52 paraffin sections, 51 staining intracellular antigens with intact cells, 52 HER2 gene amplification assay, 101–106 metallographic applications, See Autometallography; Gold cluster labels; Immunogold-silver staining mRNA ISH vs., 148–149 radioactive labels, 9 transcription regulatory factor localization, 133–145, See also Southwestern histochemistry triple labeling, 180 TSA protocols, 69–70, 75, See also Tyramide signal amplification Immunostaining, See also Immunoenzymatic methods; Immunohistochemistry (IHC) techniques; specific applications, methods, stains apoptosis markers, 202–206 consecutive sections for antigen co-localization, 1–2 double-immunofluorescence staining, 9–18, See also Immunofluorescence methods double and multiple staining techniques, See Multiple immunostaining techniques In situ end labeling (ISEL), 199, 207 In situ hybridization (ISH), 30, 147, 148 automated applications, See Automated mRNA in situ hybridization controls, 112, 196 digital optical microscope applications, 218 enzyme-labeled-fluorescence-97 protocols, 73–75 enzyme metallography, 87 evaluation of results, 279 FISH, See Fluorescence in situ hybridization fluorescence amplification for, 65–80, See also Enzyme-based fluorescence amplification; Fluorescence in situ hybridization
300
formaldehyde fixation and, 278 gold cluster labeling, 93–94, See also GOLDFISH gold enhancement protocol, 90 hybridization temperature, 154 imunohistochemistry vs., 148–149 metallographic HER2 gene amplification assay, 101–106 molecular sensitivity, 107–108 PCR, See In situ polymerase chain reaction polyclonal antibodies, 254 radioactive labels, 107 standardization, 148 transcription factor localization, 133, See also Southwestern histochemistry TSA protocols, 70–71, 75, See also Tyramide signal amplification tyramide-amplified Nanogold-silver/gold in situ hybridization, 108–120 autometallography general guidelines, 115–117 CARD protocol, 110–113 GOLDFISH protocol, 113–115 performance factors, 118–119 whole-mount, 157–166, See also Whole-mount in situ hybridization In situ polymerase chain reaction (IS-PCR), 108, 98 amplicon length, 189 applications, 179 controls, 196 DNA/RNA extraction, 180 gold cluster labeling, 93 hybridization, 196 optimization, 180 polyclonal antibodies, 254 protocols direct labeling, 195 fixation and washes, 191–193 heat treatment, 191 reverse transcription, 193–194 simultaneous multiple labeling, 195–196 thermal cyclers, 194–195 tissue preparation, 190–191 reverse transcription, 179, 186, 193–194 review of PCR technique, 181–190 thermal cycles, 194–195 triple labeling, 180 In situ 3SSSR, 108 In situ zymography, 245–251, See also Film in situ zymography Incodicarbocyanine (Cy 5), 14, 19 Indirect/direct double-staining format, 37–39, 54–56 Indirect/indirect double staining approach, 35–37, 53–54 Indocarbocyanine (Cy3), 14, 67, 95 Institutional Animal Care and Use Committee (IACUC), 265–266 Internal quality control, 280 antigen retrieval, 283–285 fixation and tissue preparation, 280–281
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Index immunocytochemical reagents, 281–283 Interphase cells, FISH applications, 168 Intracellular antigens, staining with intact cells, 52 Iron pigment, 49 ISO 9000 series standards, 281
L Laser scanning confocal microscopy, 14, 16, 91, 209, 230–231 Legal issues, animal-derived reagents, 264–269 Levamisole, 5, 48, 78 Light microscopy multiple-enzyme immunoenzymatic methods, 4–6 Nanogold and correlative EM, 91 single-enzyme immunoenzymatic methods, 2–4 three-dimensional imaging solutions, 209–222, See also Digital optical microscope Lipofuchsin, 49 Liposomes adjuvant delivery, 256 gold cluster labeling, 92–93 Liquid-based cytology, FISH applications, 169, 170–171, See also under Fluorescence in situ hybridization Locus-specific identifier (LSI) probes, 170
M Macrophages, PKC signal transduction model, 225–242, See also Signal transduction, real-time visualization TNF-α production protocol, 240–242 transfection, 226, 229–231, 233–242 Marina Blue, 15 Melanin, 49 Messenger RNA (mRNA) fluorescence amplification, 65–66, See also Enzymebased fluorescence amplification in situ hybridization applications, 147–155, See also Automated mRNA in situ hybridization; In situ hybridization precautions for analyzing, 112–113 primer design for amplification, 188–189 whole-mount ISH, See Whole-mount in situ hybridization Metallography, See Autometallography Metalloproteinase expression, in situ zymography, 245–251 Metallosomes, 92 Microarrays, 149 automated ISH applications, 152–153 comparative genomic hybridization, 123–132, See also Comparative genomic hybridization DNA isolation, 125 DNA labeling, 126–128 hybridization protocol, 128–132 troubleshooting, 132 visualization protocol, 131–132
washing protocol, 130–131 Microscope filters, 17 Microtubule visualization, 94–95 Mirror image sections, 2 Molecular-based medicine, standardization and quality assurance issues, 275–279, See also Quality assurance Molecular morphology, 107 Monoclonal antibodies animal welfare issues, 254, 258–260, 268–269 multiple immunostaining applications, 10 Mono Mac-6, 235, 237, 238, 240 Montanide ISA50, 256 Mouse macrophage transfection, 226, 229–231 mRNA, See Messenger RNA Mullis, Kary, 181 Multiple immunostaining techniques, 1–22, 27–62, See also specific applications, methods, reagents adapting immunoenzyme systems for other detectors or chromophores, 49 aims of, 29–30 antibody conjugate selection, 41–43 antibody elution, 2–6, 32 antibody tracers, 44–45 automated systems, 8 avidin-biotin complex, 7 bright-field microscopy applications, 2–9 multiple-enzyme immunoenzymatic methods, 4–6 single-enzyme immunoenzymatic methods, 2–4 CARD protocol, 20–21, See also Catalyzed reporter deposition chromogens, 4–5 co-localization, 1–2, 27–29 color combinations, 45–46, 50 antigen/color matching, 47–48 computerized imaging, 46–47 mixed color visualization, 50–51 commercial visualization systems, 59 comparison of immunofluorescence and immunoenzymatic techniques, 30 cross-reactivity, 5 DakoCytomation Animal Research Kit, 38–39, 43, 50, 56 detection systems, 43–44 direct/direct system, 34–35, 53 double-PAP staining, 6–7 endogenous enzyme activity, 48–49 film in situ zymography, 245–251 general immunoenzymatic study, 39–40, See also Immunoenzymatic methods gold particles, 8–9, See also Gold cluster labels guidelines for successful double staining, 50 IHC general protocols, 51–52 immunofluorescence techniques, 9–18, 30, See also Immunofluorescence methods avoiding cross-reactivity, 9–12 direct and indirect techniques, 9–10
301
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Molecular Morphology in Human Tissues: Techniques and Applications enzyme substrates, 17–18 fluorophores, 13–16 microscope filters, 17 immunoreagent list, 56–57 indirect/direct format, 37–39, 54–56 indirect/indirect system, 35–37, 53–54 labeled primary antibodies, 6 non-elution techniques, 6–9 order of enzymatic development, 49 PAP-combined triple immunofluorescent staining, 18 peroxidase and other label combinations, 7–9 protocols, 19–21 quadruple immunoenzymatic staining, 18 quadruple immunofluorescence staining, 19 radioactive labels, 9 requirements for successful performance, 30–32 selecting double staining concept, 41–43 sequential immunoenzymatic double staining protocol, 2, 32–34, 52–53 serial sections for multiple antigen colocalization, 1–2, 27–28 sheltering first immunoreagents, 32–33 silver particles, 9 single staining consecutive sections, 1–2 tissue pretreatment, 50–51 triple immunofluorescence staining, 20 Muramyl tripeptide phosphatidyl ethanolamine (MMPPE), 256 Myosin heavy chain mRNA, 148
N Nanogold, 82, 116, See also Gold cluster labels advantages, 85–86 autometallography general guidelines, 115–117, See also Autometallography combined fluorescent labeling applications, 94–97 digital optical microscope applications, 218 double labeling, 86 enhancement, See Autometallography IGSS, See Immunogold-silver staining immunolocalization, 85 in situ PCR, 108 microarray and biochip applications, 91–92 silver enhancement protocols, 87–89 tyramide-amplified silver/gold enhanced in situ hybridization, 108–120 CARD protocol, 110–113 GOLDFISH protocol, 113–115 performance factors, 118–119 α-Naphthol-pyronin, 4 Neuromelanin, 66 Neutral buffered formalin, 279 New Fuchsin, 45–46, 58, 59 NF-κB, 144 Northern blot analysis, 154 Nova Red, 45–46, 59
302
O OD (optical density) picture, 47 Oligo-DNA probes, 142 Oligo-histochemistry, 134, See also Southwestern histochemistry Oligonucleotide primers, 182–183, 187–190 Oncogenes, 123 Southwestern histochemistry, 144 Opsonized-fluorescent glass beads, 226–228 Oregon Green, 14, 15
P p53, 282, 288 Pacific Blue, 15 Pain treatment, 255 Paraffin section protocols, 51 in situ PCR, 190 Southwestern histochemistry, 136–137, 143 Paraformaldehyde solution, 135, 200–201 Paraformaldehyde vapor, 11, 21 pBR 322, 134 Peripheral blood leukocyte suspension, 190 Peroxidase-anti-peroxidase (PAP), 6–7, 18 Peroxidases, See also Horseradish peroxidase dextran polymer technique, 7–8 double immunostaining systems, 6–9 endogenous activity, 48 fluorogenic substrates, 18 substrates (chromogens), 4 pH antigen retrieval and, 284–285 fluorophore sensitivity, 15 gold enhancement and, 84 silver enhancement and, 83 Phage display recombinant antibody technology, 261–263 Phagocytosis, real-time PKC signal transduction visualization model, 225–242, See also Signal transduction, real-time visualization flow cytometry, 239–240 ingestion rate, 231–233 macrophage transfection protocols, 233–242 macrophages as vectors, 225–226 p-Phenylenediamine, 4, 16 Phosphate buffered saline (PBS), 135, 202 Photosensitivity, 83 Phycoerythrin (PE), 14, 35 Pit-1, 133, 134, 139, 143–144 Plant-derived antibodies, 263 Platinum labeling, 97 Polarization contrast, 210 Polyclonal antibodies animal welfare issues, 254–258 multiple immunostaining applications, 10 Polymerase chain reaction (PCR), 107, 179 amplicon length, 189 basic mechanism, 182
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Index denaturation and annealing, 183–184 extension, 184 forensic applications, 181–182 geometric amplification, 185 historical development, 181 in situ, See In situ polymerase chain reaction mRNA amplification, 188–189 oligonucleotide primers, 182–183, 187–190 reverse transcription, 185–187 thermal cycles, 183–185 Polymethylmethacrylate nanoparticles, 256 Ponceau 3R, 246, 247–248 PRE binding protein (PREB), 133, 134, 139–141, 143 Pre-market notification, 282 Prolactin gene expression, 133, 134, 141, 143–144 Protamine mRNA ISH, 152 Proteinase expression, in situ zymography, 245–251 Proteinase K, in situ PCR protocols, 193 Protein kinase C (PKC), real-time transduction visualization methods, 225–242, See also Signal transduction, real-time visualization fluorescent marker accumulation, 233 Proteolytic enzymes, formalin fixation and, 278 Pyrenyloxytrisulphonic acid derivatives, 15 Pyrocatechol, 4
Q Quadruple staining, 18–19 immunoenzymatic staining, 18 immunofluorescence staining, 19 PAP-combined triple staining, 18 Quality assurance, 275, 280–290 automated image analysis, 285–286 external quality assessment, 288–289 internal quality control, 280 antigen retrieval, 283–285 immunocytochemical reagents, 281–283 pre-market notification, 282 reproducibility of assay results, 285–287 standard reference materials, 287–288 standardization issues in molecular medicine, 275–277 antigen retrieval, 278 assays, 278–279 evaluation of results, 279–280 fixation, 289 formalin fixation, 277–278 Quantum dots, 77, 289
R Radioactive labels, 65, 107 Radioimmunohistochemistry, 9 Reagent internal quality control, 281–283 Reagent production, ethical issues, 253–270, See also Animal welfare, reagent production issues Reagent sources, 56–57, 62 Recombinant antibodies, 260–264
Red-blue color combination, 45–46, 50 Red fluorophores, 14 Red-turquoise color combination, 46 Resin removal, 202–203 Resorufin, 18 Responsive element, 133 Restauration microscope systems, 209 Reverse transcriptase (RT) enzymes, 186–187 Reverse transcriptase-polymerase chain reaction (RTPCR), 154, 181 controls, 196 in situ PCR, 179, 185–187 protocols, 193–194 Rhodamine G, 66 Rhodamine isothiocyanate (RITC), 218 Riboprobe system, 150–151 RNAases, 113, 185 RNA extraction, 180 RNA reverse transcription, 185–187, 193–194 rTth, 187, 189
S Sequential double staining techniques, 2, 32–34, 52–53 Serial sections for multi-antigen co-localization, 1–2, 27–28 Sheep red blood cells, IgG opsonization, 228–229 Signal transduction, real-time visualization, 225–242 dual parameter flow cytometry, 239–240 IgG opsonized fluorescent glass beads, 226–228 IgG opsonized sheep red blood cells, 228–229 localization time, 233 macrophage transfection, 229–231, 233–242 human and mouse cell transduction, 237–239 viral plasmid construction, 235–237 phagocytosis ingestion rate, 231–233 PKC fluorescent marker accumulation, 233 TNF-α production protocol, 240–242 viral transfection vectors, 225 Silver enhancement, 82–84, 109 combined Nanogold/fluorescent probes, 97 protocols, 87–89, 117–118 Silver particle double immunostaining system, 9 SILVERFISH, 94 Single-chain variable fragment (scFv) structures, 261–263 SNARF dyes, 15 Sodium methoxide solution, 202 Sodium thiosulfate, 117 Southwestern histochemistry, 133–145 cell culture, 137–138 controls, 142 fixation, 143 frozen tissue sections, 135–136 general guidelines and precautions, 141–142 haptenized probes, 142 heat antigen retrieval, 143 paraffin embedding, 136–137, 143 probe and salt concentrations, 142–143
303
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Molecular Morphology in Human Tissues: Techniques and Applications visualization results, 138–141 Space image microscope, 209 Specol, 256 Spectral imaging, 47 Standard reference materials, 287–288 Standardization, 275–277, See also Quality assurance assays, 278–279 automated mRNA ISH and, 148 immunocytochemical reagents, 281–283 tissue preservation, 277–278 Streptavidin-biotin complex (S-ABC), 7, 10, 20, 35, 48 Streptavidin-Nanogold, 95–96, 102, 108, 112 Streptavidin peroxidase, 119 Swiss animal welfare laws, 268–269
T TdT reaction mixture, 203 Telomeric region probes, 170 5,6,7,8-β-Tetralol carboxylic acid-β-naphthylamide phosphate, 67 Tetramethyl benzidine (TMB), 57–58, 59 Tetramethylrhodamine isothiocyanate (TRITC), 14 Texas Red, 14, 218 Thermal cycler, 183, 194–195 Thin-layer cytology, FISH applications, 167–177, See also under Fluorescence in situ hybridization ThinPrep system, 170 Three-dimensional (3D) imaging solutions, 209–222, See also Digital optical microscope immunohistochemistry, 217–218 in vivo colocalization, 210 molecular morphology, 218–219 3D-3D co-localization, 210–211, 219 Tissue dehydration, 201 Tissue fixation, See Fixation Tissue microarrays, 149, See also Microarrays Tissue transglutaminase (TTG), 199–200, 204–208 TMB (tetramethyl benzidine), 57–58, 59 Transcription factor localization, 133–145, See also Southwestern histochemistry Transfection models, real-time PKC signal transduction visualization methods, 225, 229–231, 233–242, See also Signal transduction, realtime visualization Trans-illumination technique, 210 Trastuzumab, 101, 276 Triple-CARD, 12 Triple immunostaining, See also Multiple immunostaining techniques aims of, 29–30 immunoenzymatic staining, 40 immunofluorescence staining, 20 single-PAP, 18 triple PAP, 18 Triple labeling, 180 Tubules, 205 Tumor comparative genomics, 123–132, See also Comparative genomic hybridization
304
Tumor necrosis factor-α (TNF-α), 226, 240–242 Tumors, as quality controls, 287–288 Tween 20, 44 Tyramide signal amplification (TSA), 10, 43, 67–68, 108, See also Catalyzed reporter deposition applications and results, 75 automated ISH applications, 155 combined quantum-dot detection, 77 detection methods, 76–77 enzyme-labeled-fluorescence-97 protocols, 71–75 GOLDFISH, 102, 104, 113–115 multi-labeled TSA detection, 77 Nanogold-silver/gold ISH, 108–120 CARD protocol, 110–113 GOLDFISH protocol, 113–115 performance factors, 118–119 performance factors, 119 protocols, 69–71 tyramide concentration optimization, 68
U Ultraviolet or near-UV illumination, 67 United States animal welfare laws, 265–266 U.S. Food and Drug Administration (FDA), 282
V Vaccines/vaccination, 179, 254–256 Vascular endothelial growth factor (VEGF), 151, 276 Vector Blue, 59 Vector Red, 59 Viral infections, 179 Viral plasmid construction, 235–237 Von-Willebrand factor (vWF), 158
W Washing protocol, for microarray analysis, 130–131 Water-soluble dyes, 15–16 Western mosquitofish, 97 Whole-mount in situ hybridization, 157–166 animal models, 157–161 anti-sense cRNA probe, 158, 161–162 hapten-modified probe, 157 hybridization, 162 automated protocol, 163–165 manual protocol, 162–163 manual vs. automated, 165 specimen isolation and fixation, 158–161 technical hints and discussion, 165
X X-gal, 43, 46, 49, 58–59
Z Zymography, See Film in situ zymography
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Color Figure 1.1
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Color Plate 2.1 (Figures 1–6)
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Color Plate 2.2 (Figure 7)
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Color Figure 3.1
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Color Figure 4.2
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Color Figure 5.1
Color Figure 7.1
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Color Figure 11.1
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Color Figure 12.4
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Color Figure 14.3
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Color Figure 14.4
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Color Figure 14.5
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Color Figure 14.6
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7A
7B
8A
8B
9A
9B
Color Figures 14.7–14.9
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Color Figures 14.10–14.11
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Color Figure 15.1
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Color Figure 15.2
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