Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy

Microscopy, Immunohistochemistry, and Antigen Retrieval Methods

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Microscopy, Immunohistochemistry, and Antigen Retrieval Methods For Light and Electron Microscopy

M. A. Hayat Kean University Union, New Jersey

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-47599-5 0-306-46770-4

©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©2002 Kluwer Academic/Plenum Publishers New York All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

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To my friends for their generosity

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Preface

There are several important reasons for publishing this book. One reason is to present chemical and physical principles governing the processing of tissues using microwave heating as an adjunct to fixation, embedding in a resin, and staining. A second reason is to point interested readers to a number of recent developments in the retrieval and localization of antigens in normal and pathological tissues. The greatest concentration of work in this field has focused on the detectability of disease-related proteins. Therefore, as examples, the detectability and the role in disease of estrogens, p53, p185, Ki-67, and PCNA are discussed in detail. A third reason is to review favorable aspects of the histochemical approach, whereby it yields data not obtainable by any other means, including biochemical assays. Histochemistry, for instance, contributes to acquiring knowledge about the biological activity of normal and diseased cells, which is supported by illustrations. Immunohistochemistry defines the function of cell types in a tissue and organs by localizing and identifying their contents or products. This methodology is highly visual; illustrations, especially color images, often contribute as much to correct understanding and interpretation of the results as the text. Therefore, the results of many methods are illustrated. During the last decade there has been significant progress in understanding the mechanisms responsible for antigen masking during fixation and subsequent unmasking, primarily by heating or, in some cases, by enzymatic digestion. Comparative studies demonstrate, for example, that not only microwave heating but also other sources of heating are effective in antigen retrieval. Similar studies also indicate that although sodium citrate buffer is in common use as the antigen retrieval fluid, unmasking of certain antigens requires other fluids. These and other new developments are discussed in this volume. In preparing the reader to study the location of proteins and carbohydrates, it is necessary to explain the advantages and limitations of the study. A potential limitation of the immunohistochemical approach arises from the possibility of false-negative staining due to the failure of an antibody to yield positive results. It is equally important to be aware of the possibility of false-positive staining, which can arise if the method is not scrupulous regarding histochemical negative and positive controls. Suggestions are offered to at least minimize these histological artifacts. In this regard the importance of negative and positive controls cannot be overemphasized. Negative controls involve the omission of the primary vii

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antibody with an immunoglobulin that is directed against an unrelated antigen. This immunoglobulin must be of the same class, source, and species. Positive controls involve the use of a tissue section of known positivity. The absence of staining in a test tissue section does not necessarily indicate that the antigen is not present. It should be noted that some antigens are present not only in pathological tissues but also in healthy tissues. The arrival of methods and instruments to investigate disease processes at the molecular level induces pathologists to apply these new procedures to existing problems of disease pathogenesis and disease evolution and offers clues to therapeutic intervention. Such methods include histological microdissection (in conjunction with real-time quantitative reverse transcriptase–polymerase chain reaction), cDNA microarray, anticancer vaccines, and gene regulation (genes can be turned on and off). Some of these techniques are summarized in Chapter 1. The limited space available did not allow a detailed presentation of the expanding world of molecular pathology. Chapter 1 contains seemingly diverse topics, but all are related to immunopathology. It is my hope that pathologists will benefit from these step-by-step protocols, which are presented in a self-explanatory form so that the reader can practice them without outside help. Chapter 8 contains details of specific methods because the various parameters of processing of each type of antibody, antigen, and tissue may need to be varied to obtain optimal results. I have tried to synthesize a large number and variety of immunohistochemical techniques into a single and concise basic handbook. Some alternative methods are also included. I have explained not only how to use a technique but also why to use it—along with its advantages and limitations. An example is the microwave heating methodology. The methods presented were carefully selected and are reproducible but can be modified, depending on the objective of the study. Moreover, as the antigen retrieval methodology is relatively new, it requires fine-tuning. There is a degree of necessary repetition in some chapters, which allows them to stand alone. This approach helps the reader to carry out a procedure where it is described without searching for its details somewhere else in the book. Cross-reference of information among chapters, wherever possible, is given. Where possible, commercial sources of reagents, kits, and equipment are listed throughout the text instead of in a separate index. Extensive references are provided to facilitate the task for those readers who may wish to consult the literature for additional information on specific topics. All books can be improved, and this volume is no exception. I welcome constructive criticism from my colleagues and students. With this help I look forward to offering a greatly improved second edition. The writing of this book would not have been possible without the most generous help of a large number of distinguished scientists. I am very grateful for the thoughtful and invaluable suggestions and illustrations received from scientists throughout the world. It is appropriate to acknowledge significant contributions made to the understanding and practice of antigen retrieval methodology and applications of microwave heating by Hector Battifora, Mathilde E. Boon, Giorgio Cattoretti, Richard J. Cote, Ann M. Dvorak, Jules M. Elias, Johannes Gerdes, David Hopwood, Allen M. Gown, Richard Horobin, L. P. Kok, Anthony S.-Y. Leong, Gary R. Login, Enrico Marani, Shan-Rong Shi, Albert J. H. Suurmeijer, and Clive R. Taylor. It is not possible to mention all the scientists who have played a role in the development of this technology.

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The help and encouragement received from Dean Betty Barber throughout the writing of this book are greatly appreciated and will be remembered. I thank Patricia Lemus and Elizabeth McGovern for their expert secretarial assistance in the preparation of the manuscript, and I appreciate the help and cooperation extended to me by Roberta Klarreich, the production editor, throughout the production of this volume. M. A. Hayat October 2001

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Contents

Chapter 1. Introduction Cytogenetic Evaluation of Tumors Genetic Instability of Tumors Tumor Heterogeneity Histological Microdissection

Antitumor Vaccines Molecular Genetics cDNA Microarray Technology Angiogenesis Vascular Endothelial Growth Factor Immunohistochemical Localization of Vascular Endothelial Growth Factor Telepathology (Telemedicine)

Future of Immunohistopathology Preparation of Buffers

Chapter 2. Antigens and Antibodies Antigens Epitopes

Antibodies Polyclonal Antibodies Production of Polyclonal Antiserum Affinity Chromatography Monoclonal Antibodies Specificity of Monoclonal Antibodies MIB-1 Monoclonal Antibody Production of Monoclonal Antibodies Bivalent and Bispecific Monoclonal Antibodies in Cancer Therapy

Recombinant Antibodies Anticancer Monoclonal Antibodies

1 11 12 14 14 15 17 18 20 23 24 25 28 29

31 31 32 33 34 35 35 37 38 39 41 44 46 47 xi

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Antibody Cross-Reactivity Polyreactive Antibodies Commercial Sources of Antibodies

48 49 50

Chapter 3. Fixation and Embedding

53

Formaldehyde

53 54 54 56 57 58 59 60 61 62 64

Nature of Formaldehyde Solution Mechanism of Fixation with Formaldehyde Comparison of Formaldehyde with Glutaraldehyde Fixation with Formaldehyde Effect of Prolonged Fixation with Formaldehyde Formalin Substitute Fixatives Fixation Conditions

Effect of Heating on Fixation with Glutaraldehyde Microwave Heat–Assisted Fixation with Osmium Tetroxide Role of Microwave Heating in Enzyme Cytochemistry Fixation for Enzyme Cytochemistry Using Microwave at Relatively Low Temperature

Cryopreservation in the Presence of Microwave Heating Paraffin Embedding Paraffin Embedding in Microwave Oven Paraffin Embedding in Vacuum-Microwave Oven Microtomy of Paraffin-Embedded Tissues Silanting of Glass Slides Vacuum-Assisted Microwave Heating

Chapter 4. Factors Affecting Antigen Retrieval Fixation Denaturation Heating pH Molarity Antigen Retrieval Fluids Glycerin as Antigen Retrieval Fluid Procedure pH of Antigen Retrieval Fluids Ionic Strength of Antigen Retrieval Fluids

Antibody Penetration Antibody Dilution Diluent Buffer for Primary Antibodies

Storage of Paraffin-Embedded Tissues Storage of Tissue Slides Signal Amplification

64 65 65 67 67 67 68 69

71 71 72 73 74 74 75 77 78 78 79 79 80 82 83 84 89

Contents

xiii Tyramine Amplification Method Preparation of Biotinylated Tyramine Rolling Circle Amplification

Chapter 5. Problems in Antigen Retrieval Lack of Immunostaining Background Staining Problem of Endogenous Biotin Procedure

Mirror Image Complementary Antibodies Procedure

Fixation of Frozen Tissues Hot Spots (Areas) in Microwave Oven Problem of Antigen Retrieval Standardization Test Battery Intraobserver and Interobserver Variation in Diagnosis Quantitation of Immunostaining Autostainers Capillary Gap Stainers Centrifugal Stainers Flat-Method Stainers

Volume-Corrected Mitotic Index The Gleason Grading System Universal Antigen Retrieval Method? Calibration of Microwave Oven

Chapter 6. Antigen Retrieval Possible Mechanisms of Antigen Retrieval Nonthermal Effects of Microwave Heating

Effect of Endogenous Calcium on Antigen Masking Use of Ethylenediaminetetraacetic Acid (EDTA) for Antigen Retrieval Antigen Retrieval with Heat Treatment Advantages of Heating Heating Methods Mechanism of Epitope Retrieval by Microwave Heating Duration of Microwave Heating Antigen Retrieval in a High-Pressure Microwave Oven Antigen Retrieval at Low Temperature Use of Heat for Staining Rapid Immunostaining of Frozen Sections Enhanced Polymer One-Step Staining Procedure Modified En Vision Procedure Hazards and Precautions in the Use of Microwave Ovens

90 92 92

95 95 96 98 100 101 101 102 102 103 104 105 105 107 109 109 109 110 111 113 114

117 117 119 120 123 124 124 125 130 131 132 132 136 138 139 139 141

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Limitations of Microwave Heating

Wet Autoclave Method Procedure 1 Procedure 2

Ultrasound Treatment Procedure

Nonheating Methods Detergents Procedures Proteolytic Enzyme Digestion Procedure Enzyme Digestion and Relatively Low Temperature (80°C)–Assisted Antigen Retrieval

Comparison of Antigen Retrieval Methods: A Summary

Chapter 7. Antigen Retrieval on Resin Sections .................... Role of Fixative and Embedding Resin in Antigen Retrieval Immunostaining of Thin Resin Sections Antigen Retrieval on Sections of Modified Epoxy Resin Effect of Heating Antigen Retrieval on Thin Resin Sections Using Autoclaving Rapid Staining of Thin Resin Sections in Microwave Oven Microwave Heat–Assisted Rapid Processing of Tissues for Electron Microscopy Microwave Heat–Assisted Immunolabeling of Resin-Embedded Sections Microwave Heat–Assisted Immunogold Methods

142 145 145 146 146 148 148 148 149 151 152 152 153

155 156 158 160 161 161 163 163

Immunogold-Silver Staining Droplet Procedure

163 167 167 167

Chapter 8. General Methods of Antigen Retrieval

169

General Procedure for Antigen Retrieval Using Microwave Heating Antigen Retrieval in Archival Tissues Method for Microwave Heating of Archival Tissue Blocks

Antigen Retrieval Using a Conventional Oven Hot Plate–Assisted Antigen Retrieval Procedure

Hot Plate–Assisted Grading of Vulvar Intraepithelial Neoplasia Water Bath Heat–Assisted Antigen Retrieval Procedure for Electron Microscopy Procedure for Light Microscopy Procedure for Free-Floating Sections

Microwave Heat–Assisted Evaluation of Global DNA Hypomethylation

169 173 174 175 175 175 176 177 178 178 180 180

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Procedure

Microwave Heat–Assisted Enhanced Peroxidase One-Step Method Procedure

Microwave Heat–Assisted Immunostaining of Cell Smears Double Immunostaining Using Microwave Heating Microwave Heat–Enhanced Double Immunostaining of Nuclear and Cytoplasmic Antigens Procedure

Microwave Heat–Assisted Immunohistochemical Localization of Cyclin D1 Microwave Heat–Assisted Immunofluorescence Staining of Tissue Sections Procedure

Microwave Heat–Assisted Double Immunofluorescence Labeling Procedure

Microwave Heat–Assisted Double Indirect Immunofluorescence Staining Procedure Control Procedures Immunoenzymatic Detection

Combined Microwave Heating and Ultrasound Antigen Retrieval Method Combined Enzyme Digestion and Microwave Heating Antigen Retrieval Method Pressure Cooker–EDTA–Assisted Antigen Retrieval 2-Mercaptoethanol–Sodium Iodoacetate–Assisted Antigen Retrieval Antigen Retrieval with Steam–EDTA–Protease Method Procedure

Picric Acid–Steam Autoclaving–Formic Acid–Guanidine Thiocyanate–Assisted Retrieval of Prion Protein Procedure

Simultaneous Detection of Multiple Antigens Procedure

Use of Multiple Antibodies for Labeling Antigens Procedure

Antigen Retrieval in Neuronal Tissue Slices before Vibratome Sectioning Microwave Heat–Assisted Antigen Retrieval in Freshly Frozen Brain Tissue Procedure

Microwave Heat–Assisted Rapid Immunostaining of Frozen Sections Procedure

Microwave Heat–Assisted Immunocytochemistry of Thin Cryosections Procedure

181 181 181 182 182 183 183 184 185 186 186 186 187 187 188 189 189 190 191 191 192 192 192 194 194 196 196 197 198 198 199 199 200 200 201

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Pressure Cooker–Assisted Detection of Apoptotic Cells Immunohistochemical Localization of Prostate-Specific Antigen Immunohistochemistry Procedure

Chapter 9.

Other Applications of Microwave Heating Carbohydrate Antigens Ovarian Carcinoma Microwave Heat–Assisted Carbohydrate Antigen Retrieval Enzyme Digestion–Assisted Carbohydrate Antigen Retrieval

Nucleolar Organizer–Associated Region Proteins Procedure Nucleolar Size

In Situ Hybridization Radioactive Probes Nonradioactive Probes Enhancement of in Situ Hybridization Signal with Microwave Heating

Procedure for in Situ Hybridization of DNA In Situ Hybridization of RNA in Skeletal Tissues Microwave Heating for in Situ Hybridization of mRNA in Plant Tissues Microwave Treatment Staining Microwave Heat–Assisted Fluorescence in Situ Hybridization Procedure for Gastrointestinal Neoplasia Nuclear Fluorescence in Situ Hybridization Signal Using Microwave Heating

201 202 203 203

205 205 206 208 208 209 211 212 213 215 215 217 218 219 221 221 221 222 222

Procedure

223 224 224 225 225 227 228 228 229 230 230 230

Chapter 10. Cell Proliferating Antigens

233

Ki-67 Antigen

233 235 237 237

Microwave Heat–Assisted Polymerase Chain Reaction Procedure

Detection of Antigens by Flow Cytometry Microwave Heat–Assisted Flow Cytometry Procedure 1 Procedure 2

Microwave Heat–Assisted Microwave Heat–Assisted Microwave Heat–Assisted Microwave Heat–Assisted

Enzyme-Linked Immunosorbent Assay Scanning Electron Microscopy Confocal Scanning Microscopy Correlative Microscopy

Immunohistochemistry Limitations of Immunohistochemistry Antibodies

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Contents Recent Applications of MIB-1 Antibody Ki-67 Antigen Retrieval Using Microwave Heating Ki-67 Antigen Retrieval Using Autoclave Treatment

Proliferating Cell Nuclear Antigen Immunohistochemistry Limitations of PCNA Immunohistochemistry Immunostaining of PCNA on Cryostat Sections

p53 Antigen Wild-Type p53 Protein Mutant p53 Protein p73 Antibodies Examples of Antibody Dilutions Immunohistochemistry

Use of Multiple Antibodies for Labeling p53 Antigen Wild-Type p53 Antigen Retrieval Using Microwave Heating p53 Antigen Retrieval Using Microwave Heating Frozen Section Immunohistochemistry of p53

Chapter 11. Estrogens Estrogen Receptors Estrogen Receptor Alpha Estrogen Receptor Beta Estrogen Receptor Gamma Distribution of Estrogen Receptors

Role of Estrogen Receptors in Breast Cancer Breast Cancer and Tamoxifen

Antibodies Immunohistochemistry Comparison of Immunohistochemistry with Biochemical Ligand-Binding Assays Dextran-Coated Charcoal Assay

Semiquantitative Assessment of Estrogen Receptors Immunostaining of Estrogen Receptors in Prostate Tissue Immunostaining of Estrogen and Progesterone Receptors in Fine-Needle Aspirates of Breast

Chapter 12. HER-2 (c-erbB-2) Oncoprotein HER-2/neu Oncogene HER-2 Oncoprotein HER-2 Overexpression Simultaneous Overexpression of HER-2 and p53 Distribution of HER-2 in Carcinomas

239 240 240 241 242 243 245 245 247 248 249 250 253 253 256 257 257 258

261 262 265 267 268 268 269 270 270 273 275 276 276 277 278

281 281 283 284 284 285

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Contents Astrocytic Tumors Bladder Carcinoma Ewing’s Sarcoma Intrahepatic Cholangiocellular Carcinoma Laryngeal Squamous Cell Carcinoma Non-Small-Cell Lung Carcinoma Ovarian Carcinoma Prostate Carcinoma Squamous Cell Carcinoma of Cervix

Methods for Detecting HER-2 Status Quantitative Analysis of HER-2/neu Gene Expression Detection of HER-2 Oncoprotein Bispecific Antibodies Bispecific Antibody MDX-H210

Vaccines Genetic Immunization

Immunohistochemistry Herceptin (Trastuzumab) HercepTest Controls and Scoring System

Immunostaining of HER-2 Protein Using HercepTest

285 285 285 286 286 286 287 288 288 289 290 291 293 294 295 295 296 297 299 300 302

References

305

Index

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Microscopy, Immunohistochemistry, and Antigen Retrieval Methods

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Chapter 1

Introduction

Immunohistochemistry and immunocytochemistry have played an important role in the fields of cell and tissue biology, embryology, and diagnostic pathology. These methodologies facilitate precise analysis of the chemistry of cells and tissues in relation to structural organization. The information derived from these techniques will continue to contribute to our understanding of dynamic molecular, cellular, and pathological processes. For example, immunohistochemistry has revolutionized the field of tumor diagnosis and has provided a powerful tool for pathologists to better characterize difficult or unusual neoplasms. In addition, this technology has provided information that has resulted in the reclassification of many neoplasms and, in some cases, the creation of new categories that were previously unrecognized. In this volume the emphasis is on the application of this methodology to routine diagnostic pathology. The immunohistochemical method localizes and identifies a specific antigen in a cell or a tissue specimen. In the most common approach, specimens are fixed in 10% neutral buffered formalin and embedded in paraffin. Sections ( thick) are deparaffinized with xylene and then rehydrated in a series of ethanol solutions with decreasing concentrations. After drying, sections are treated with 3% hydrogen peroxide to block endogenous peroxidase. If required, sections are subjected to antigen retrieval. Nonspecific immunostaining is blocked by treating the sections with 10% normal serum. The primary antibody applied is either monoclonal or polyclonal. A secondary antibody, linked with an imaging system (usually a peroxidase), is applied to recognize the primary antibody. Contrary to the popular use of the term antigen retrieval in the literature and by commercial companies, the correct term is epitope retrieval, since it is the epitope (antigenic determinant) that is recognized by the antibody (paratope) instead of antigen molecule as a whole. A monoclonal antibody reacts with a specific region of the antigen irrespective of the conformation of other regions of the same antigen. Different monoclonal antibodies generally react with different epitopes of the same antigen. Thus, the epitope is the part of the antigen to which the antibody is directed. An epitope indeed defines antigen specificity. Furthermore, the fact that monoclonal antibodies to different epitopes of the same antigen molecule behave differently in “antigen retrieval” indicates that what is being retrieved is an epitope. In other words, two different epitopes of the same antigen may require different treatments for their accessibility to the respective antibodies. This is the context in which this subject should be understood. 1

2

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The other point of view, which favors the term antigen retrieval instead of epitope retrieval, is as follows (S.-R. Shi, personal communication). It is likely that the mechanism of antigen retrieval is based on chemical modification of protein conformation. Therefore, retrieval of formalin-modified (or masked) antigenicity must be a restoration of the protein structure, as any antigen/antibody recognition is dependent on protein conformation. This is particularly true for discontinuous epitopes (most antigen determinants are discontinuous epitopes), which consist of amino acid sequences apart from each other on one polypeptide (or actually located on distinct polypeptides) but brought near each other in the tertiary or quaternary structure of the protein. In other words, restoration of the function of an epitope (antigenicity) is the retrieval of its protein conformation, i.e., retrieval of antigen. Because the concept of epitope is not an intrinsic feature of a protein existing independently of its paratope partner, the term epitope refers to only a functional unit, but not a stoic structure of the protein. Shi et al. (2000a) have further justified the use of the term antigen and rejected the relevancy of the term epitope in immunohistochemistry. In support of Dr. Shi’s opinion is the fact that in some cases the absolute specificity of even monoclonal antibodies can be questioned. The absorption control cannot always determine whether the protein bound in the tissue is the same protein used for absorption. The monoclonal antibody may instead recognize a similar epitope of an unrelated protein, especially following tissue fixation. Absorption controls therefore may not provide the specificity of the antibody for a protein under study in the tissue. In light of the above-mentioned difference of opinion, and in the absence of a definite understanding regarding unmasking of an epitope or whole antigen molecule as a result of unmasking treatments (heat or nonheat treatment), both terms, epitopes and antigens, are used in this volume. Immunohistochemistry has surpassed other techniques in its effectiveness in the in situ preservation and detection of antigens. Immunopathology has become a valuable or even an essential adjunct to diagnostic pathology. It is affirmed that diagnostic immunohistochemistry is indispensable in surgical pathology for diagnosis, therapy, and prognosis. The usefulness of this methodology depends and will continue to rely on three major factors: (1) availability of specific primary antibodies; (2) an efficient detection system; and (3) correct interpretation and significance of the findings. An increasing number of monoclonal antibodies is being produced, and many of them are commercially available; these sources are given in Chapter 2. The role of antibodies in diagnostic pathology is discussed in this chapter. Highly sensitive detection systems are available, and their signals are being continuously enhanced to achieve high signal-to-noise ratio. Methods of scoring the signals are also available. These improvements are discussed in Chapter 4. All immunohistological methods depend on the successful completion of a series of sequential steps, beginning with specimen collection; their morphological and antigenic preservation (chemical fixation or rapid freezing) or antigenic retrieval; incubation in an antibody or sequence of antibodies; staining; signal counting; and interpretation of results. Each of these steps must be performed as efficiently and correctly as possible. If even one of these steps is suboptimal, the remaining steps, even though perfectly carried out, can never compensate for the inefficient step. Any error in immunohistochemistry, especially when applied to clinical diagnosis, is unacceptable. This methodology is not only a science but also an art; each aspect depends on the other. Progress in molecular biology is intimately related to advancement in technology. One fundamental goal of cell research is to understand the functions of molecules that

Introduction

3

constitute cells and tissues. This understanding can be enhanced by examining the molecular details and subcellular location of cell components. The precise extracellular and intracellular localization of molecules under different physiological and pathological conditions yields clues to their possible functions. These aspects of antigen molecules and receptors, especially clinically important ones such as p53, Ki-67, PCNA, p185, and estrogens, are discussed in detail in Chapters 10, 11, and 12. The achievement of the above-mentioned goal received an impetus from the development of the heating methodology (especially microwave heating) for antigen retrieval. Microwave heating was introduced into biomedical research approximately two decades ago for the rapid processing of plant and animal tissues. The development of this technique was a significant step forward in the application of histochemistry, immunohistochemistry, and immunocytochemistry. In other words, this methodology has significantly contributed to the localization of macromolecules and molecules (including antigens) and thus to an understanding of their functions. This technique is also useful for enhancing the detection of RNA and DNA by in situ hybridization (see page 213). Another example of the application of microwave heating is in conjunction with flow cytometry (see page 225). The polymerase chain reaction (PCR) has also been used in conjunction with microwave heating for studying DNA (see page 224). Yet another application of microwave heating is with the enzyme-linked-immunosorbent assay method (ELISA) (see page 228). Tissue cryopreservation with diminished ice crystal growth has also been accomplished with this versatile technology (see page 65). Application of microwave heating to enzyme cytochemistry, autoradiography, and X-ray microanalysis has been attempted (Mizuhira and Hasegawa, 1996). Significant aspects of the basic biology of disease processes are now assuming clinical importance in diagnosis and prognosis. The pathologist can identify an ever-increasing range of antigens in tissue sections using the techniques mentioned above. Identification of tissue antigens using these methods is of fundamental importance for clarifying tumor proteins or carbohydrates, determining the diagnosis and prognosis of tumors, characterizing pervasive nepotistic alterations in tissues such as prostate, subclassifying neoplasms, evaluating the response of tumors and pervasive nepotistic changes to certain therapies (i.e., as a surrogate intermediate and end point), selecting patients who are candidates for specific therapies (e.g., immunotherapy), and identifying pathogenic organisms (Arnold et al., 1996). For these and other reasons, immunohistochemistry has become the most important tool in research and diagnostic pathology. It permits detection of defined antigens on cryostat, paraffin, and resin sections of normal and diseased tissues. Immunohistochemical and immunocytochemical localization of antigens is a powerful tool that provides insight into some of the salient features of cell and tissue complexity. Such studies, for example, demonstrate relationships between normal cell structure and function and pathological consequences. Presently, immunohistochemistry is firmly established as the most important method for detecting antigens with the light microscope. It can be effectively used to examine various antigens in the sections of formaldehyde-fixed and paraffin-embedded tissues (Hayat, 2000a). The availability of the equipment to carry out immunohistochemistry and the introduction of many new monoclonal antibodies make it possible to apply this technique to retrospective studies. The introduction of a large number of new monoclonal antibodies of improved sensitivity and specificity, which are available in ready-to-use kits, has made possible a wider use of immunohistochemistry for antigen analysis. In addition, the development of various

4

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antigen retrieval methods during the past two decades has enabled many more antibodies to access antigens that were undetectable or minimally detectable in the past. Today almost any antigen that survives tissue processing has the potential to be localized immunohistochemically. As a result of these methods, additional antibodies have become paraffin- and resin-compatible, which permits heat-treated tissue sections to be used for detecting antigens with the light and electron microscopes. New antigen retrieval methods, especially microwave heating and other heating procedures and ultrasound treatment, can effectively retrieve antigens from tissues left in formaldehyde for prolonged periods. The introduction of the computer-assisted image analyzer and other automated equipment (e.g., the automatic stainer), and generation of antibodies to synthetic peptides, have ushered immunohistochemistry into a higher level of efficiency, accuracy, and quantitation. The demand for a more precise spatial localization of epitopes favors the use of antibody fragments (e.g., Fab), peptides, or ligands. These advances facilitate the use of antigen detection for correct diagnosis and prognosis. Furthermore, advances in detection accuracy provide guidelines to study and understand more complicated biological problems. To achieve these goals, standardization of antigen retrieval methods is necessary, at the least, to minimize inter- and intralaboratory (including interobserver) variability of immunostaining (see Chapter 5). However, even in the absence of such standardization, the method has become the most effective tool in light microscope immunohistochemistry and, to some extent, in electron microscope immunocytochemistry.

Introduction

5

Although antigen retrieval is carried out most commonly on paraffin sections, it can also be accomplished on semithin or thin resin sections for light and electron microscopy, respectively. Thin sections of routinely used resins such as epoxy, LR White, LR Gold, and Lowicryls can be used for detecting antigens with the light or electron microscope. These resins, in conjunction with microwave heating, can also be used for cell and ultrastructural studies with the light microscope, and scanning and transmission electron microscope (Fig. 1.1). This procedure can also be employed for studying bacteria with the scanning electron microscope (Fig. 1.2). The advantages of resin sections include better preservation of cellular details, assisting the achievement of higher resolution, and the ability to carry out correlative studies of the same tissue with the light microscope and scanning and transmission electron microscope (Fig. 1.3). In addition, resin sections (also cryosections) allow immunogold and silver-enhanced immunogold staining. Biochemical assays such as the dextran-coated charcoal (DCC) assay, certain signal amplification techniques, and other cytosol-based methods have been mostly replaced

6

Chapter 1

by immunohistochemistry because the former methods are costly and often difficult to reproduce. For example, the DCC assay requires rather large tissue specimens and may be adversely influenced by tissue heterogeneity (e.g., tumors), presence of bound endogenous estrogen, and sampling error (Hendricks and Wilkinson, 1993). In cytosol-based biochemical assays, tissues are indiscriminately homogenized (e.g., tumor, stroma, inflammatory cells, and epithelial cells). Therefore, the results expressed in fentomoles per milligram of the total protein are variably diluted because of the presence of nontumorous cells. In contrast, immunohistochemistry can be carried out with smaller tissue specimens and is less affected by tissue heterogeneity or endogenous hormones. This technique allows direct histological visualization, which permits separation of tumors from stroma, inflammatory cells, and normal cells. However, the DCC assay can be employed for correlative studies; therefore, the selection of a particular method in a diagnostic laboratory should depend on a number of factors, including specificity, sensitivity, rapidity, use of potentially harmful reagents, availability of equipment, cost, and application to a wide range of antigens. However, the central problem in immunohistochemistry is to retain antigenicity without sacrificing the quality of cell morphological preservation. It has been established that the preservation of antigenicity is inversely related to the preservation of cell morphology (Hayat, 2000a); thus, tissue preparation methods optimal for the preservation of cell morphology introduce protein crosslinking and are therefore suboptimal for preserving antigenicity. Because preserving antigenicity in immunohistochemistry is more important than preserving morphology, 10% formaldehyde is generally used for fixation. Glutaraldehyde, on the other hand, yields excellent ultrastructural preservation but severely masks most antigens by introducing irreversible protein crosslinking. The mechanism(s) responsible

Introduction

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8

Chapter 1

for the antigen-masking effects of fixatives are discussed in Chapter 4. Tissue embedding in paraffin also adversely affects antigen detection. The alternative to chemical fixation and paraffin embedding is to use frozen sections of fresh-frozen tissues (snap freezing). These cryostat sections tend to provide improved antigen detection. Proteins are retained in these sections at least until the cryosections are placed into aqueous incubation and staining solutions. Most antibodies recognize antigens better in frozen sections than in sections of chemically fixed and paraffin-embedded tissues. Fixation of frozen sections has also been recommended by some workers to immobilize proteins during subsequent processing of cryostat sections and thus improve the antigen localization. However, an agreement on the beneficial effect of this practice is lacking. Moreover, cryostat sections are difficult to prepare, and the quality of morphological preservation is comparatively poor (Fig. 1.4). Fortunately, methods are available that satisfactorily preserve cell morphology as well as antigenicity. The best compromise is to use a mixture of 6% formaldehyde and glutaraldehyde of a low concentration (0.1–0.4%). This approach, which is used extensively for electron microscopy, should be used for light microscopy and is presented in this volume. The optimal immunohistochemical method should ideally detect all specific epitopes, but in practice this is not possible. The best that can be accomplished is to optimize the protocol to detect all the detectable epitopes. Presently, the majority of the antigen retrieval studies are carried out using microwave heating, although other heating methods such as autoclaving (page 145) or pressure cooking (pages 127–128) can be equally effective, depending on the types of tissue and antigen under study. Antigen retrieval can also be accomplished in a microwave oven under vacuum, as can rapid fixation, resin embedding, and staining. Ultrafast (milliseconds) or fast (seconds to minutes) fixation can be achieved with this method. Even rapid fixation with osmium tetroxide can be carried out in a microwave oven (see Chapter 3). Like any other technique, microwave heating has certain limitations. A well-known example is the uneven distribution of hot and cold spots in the oven, although this can be minimized by using a water load in the oven. Prior to placing the water load in the oven, cold and hot spots should be located in the oven cavity by using a high-brightness neon bulb array. Hot spots must be avoided because an excessive increase in temperature during irradiation is a major cause of poor fixation. The problem of hot spots apparently can be avoided by using heating methods other than microwave heating. One should also be aware that microwave heating may promote cross-reactivity by producing or unmasking antigens closely related to those under study. The result may be the decreased specificity of an established and generally available antibody (Alexander and Dayal, 1997). Other limitations of microwave heating are listed on pages 142-144. Antigen retrieval can also be accomplished by treating paraffin sections with digestive enzymes. A number of proteolytic enzymes, including trypsin, pepsin, pronase, and ficin (a plant enzyme), have been used for unmasking antigens. However, effectiveness of each enzyme is limited to a few types of antigens. Moreover, enzymatic digestion is ineffective for antigen retrieved in overfixed tissues and tends to damage cell morphology, especially when the treatment is prolonged. Cumulative evidence indicates that heating is generally better than digestion. Therefore, the latter approach is not preferred unless the former is unsuccessful. Better results are obtained in some cases when enzyme digestion is used in conjunction with heat treatment.

Introduction

9

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Immunohistological diagnosis is critically linked to an assessment of the morphological appearance of the cell. This is accomplished by employing a panel of monoclonal antibodies to establish the immunoprofile of a tumor and, it is hoped, to minimize the risk of false-negative or false-positive staining. Therefore, a thorough knowledge of the chemistry of tissue processing, including antigen retrieval, is essential for routine practice. The antibodies described in this volume are immunoreactive in fixed, paraffin-embedded tissue sections and therefore are the mainstay of routine diagnostic histopathology. However, it should be noted that the specificity of certain monoclonal antibodies, as commonly understood, is not valid because such an antibody shows cross-reactivity, that is, binding to epitopes shared by related proteins in the same tissue or from different tissues. Finally, the use of antibodies to clinically important antigens for diagnostic and prognosis purposes requires a complete understanding of the role of antigens in the biology of disease. For correct interpretation of histological findings, the pathologist must be knowledgeable about pathophysiology, notwithstanding the availability of the most specific panel of monoclonal antibodies. For this and other reasons the role of several antigens, in health and disease is discussed in detail in Chapters 10, 11, and 12. The following discussion summarizes the role of estrogens, p53, Ki-67, PCNA, and p185 proteins in health and disease. The importance of estrogens in health and disease becomes apparent when one considers that these hormones trigger and govern essential functions such as growth, differentiation, and the functioning of many target tissues. They also significantly influence the proliferative and metastatic states of breast cancer cells. It is also known that estrogens affect the regulation of gene transcription through interaction with at least two estrogen receptors ( and ). The role of estrogens and their receptors in breast cancer is emphasized in this volume. As examples, immunohistochemical localization of these receptors in the prostate tissue and in fine-needle aspirates of breast is presented. The tumor suppressor gene p53 encodes a nuclear phosphoprotein which is expressed in most, if not all, tissues of the body. The steady-state levels of this protein in normal somatic cells are usually very small because this newly synthesized protein is highly sensitive to ubiquitin/proteasome-mediated degradation, preventing its accumulation in cells. It functions primarily as a transcription factor that is activated in response to genotoxic stress, including DNA damage. Thus it controls the expression of many genes involved in regulating the cell cycle and apoptosis. In this way, p53 prevents the excessive accumulation of mutations and harmful cells which could give rise to malignancies. On the other hand, mutation of p53 occurs frequently in human oncogenes. These mutations in tumors abrogate the regulatory function of p53 on the cell cycle and lead to increased half-life of the otherwise very unstable wild-type p53 protein. The importance of immunohistochemical localization of p53 protein becomes obvious when considering that approximately 50% of all human malignancies exhibit mutation and aberrant expression of this protein, making it an important target candidate for cancer immunotherapy. Details of immunohistochemical localization of wild-type p53 and mutant p53 are explained in this volume. Ki-67 is a nuclear and nucleolar phosphoprotein. Although the biological function of Ki-67 has not been fully elucidated, it is accepted that this antigen, along with p53, PCNA, cyclin Dl, and bc12, plays an important role in regulating somatic cell proliferation. Immunohistochemical examination using a Ki-67 labeling index is a promising proliferation marker, as a higher rate of Ki-67-positive cells correspond to greater malignancy.

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Monoclonal antibody MIB-1 is used to recognize this antigen, and so the usefulness of this antibody is discussed in detail in Chapter 2. Immunohistochemical methods using microwave heating or autoclave treatment for localizing Ki-67 are presented. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase 8 and is intimately associated with DNA replication. Indeed, direct interaction between DNA polymerase and its processivity factor PCNA is essential for effective replication of the eukaryotic genome. This protein also plays a key role in other functions, such as nucleotide excision repair, mismatch repair, base excision repair, cell cycle control, apoptosis, and transcription. These interactions support the concept that PCNA plays a central role in connecting all these important cellular processes and can function as cellular communicator in cells. Clinically useful activity of PCNA can be identified by immunohistochemistry and flow cytometry. Expression of this antigen in a cell population equates to the growth fraction, that is, the proportion of cells involved in an active cell cycle. Because PCNA is expressed in all cycling cells, the entire proportion of dividing cells present at any instant in a population can be detected. Details of immunohistochemical staining of PCNA on cryostat and paraffin-embedded sections are presented in this volume. In recent years evidence has increasingly demonstrated the importance of protooncogenes in the pathogenesis of diseases, including breast cancer. Amplification of HER-2/neu gene is found in ~25% of human breast cancers and results in the overexpression of p185 oncoprotein. Amplification of the gene in breast cancer patients is correlated with shorter disease-free states and poorer overall survival rates than in patients showing no such amplification. Accurate detection of the gene amplification in breast cancer tissues is important in determining patient prognosis as well as response to standard chemotherapeutic agents. Moreover, it is currently the sole criterion for selecting patients for HER-2/neu-targeted therapy with the recombinant humanized anti-p185 antibody Herceptin (trastuzumab) (Pauletti et al., 2000). Overexpression of this protein in breast cancer is associated with adverse prognostic factors that include advanced pathological stage, number of metastatic axillary lymph nodes, absence of estrogen and progesterone receptors, increased S-phase fraction, DNA ploidy, and high nuclear grade. Because the gene product is ultimately responsible for the biological activity of the gene, it is apparent that direct measurement of the protein or immunohistochemical analysis is as clinically relevant as is the determination of the number of gene copies (Battifora et al., 1991). Immunohistochemistry is superior to biochemical assays because it eliminates the dilution effect caused by variable amounts of stroma and other nonneoplastic tissues. Another advantage of immunohistochemistry is its ability to detect overproduction of an oncoprotein resulting from a mechanism other than gene amplification. Amplification of HER-2/neu gene and Overexpression of p185 are also found in many tissues other than those with breast cancer. Immunohistochemical detection of p185 is presented in Chapter 12.

CYTOGENETIC EVALUATION OF TUMORS A brief discussion on the usefulness of cytogenetic evaluation of tumors is in order. The diagnosis of tumors on the basis of information obtained from immunohistochemical staining alone on occasion may pose a challenge to the pathologist. Cytogenetics, which is another approach to determine the histogenetic origin of some tumors and to identify

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sites of gene deregulation for molecular analysis, can provide an important adjunct to diagnostic surgical pathology. For example, karyotypic analyses are helpful in the differential diagnosis of histologically similar small round cell tumors, including lymphoma and neuroblastoma (Sreekantaiah et al., 1994). These tumors are composed of primitive cells that often lack distinguishing features. Each of these tumors contains specific chromosome changes; thus, cytogenetic analysis provides a reliable approach that can distinguish between these neoplasms. In addition, the identification of diagnostic chromosome translocations in histologically undifferentiated tumors may support a diagnosis that is doubtful on histological grounds alone or may even lead to a reconsideration of the histological diagnosis. This approach can aid in directing therapy, determining prognosis, and identifying sites of gene perturbation for molecular characterization. Unfortunately, cytogenetic evaluation of tumors is still a relatively underutilized approach. However, new potentially promising tumor markers have been introduced based on the molecular genetic cancer research. Various genetic alterations important in carcinogenesis, of which alterations in the ras oncogenes and the p53 tumor suppressor gene are the most common, have now been described. Both of these are useful targets for diagnostic purposes. This is substantiated by considering that p53 alterations are among the most frequent genetic alterations in human malignancies. Similarly, clinical application of ras gene mutation, for example in the diagnosis of pancreatic adenocarcinoma, has been well established (e.g., Berthelemy et al., 1995). Chromosomal abnormalities in many tumors and their diagnostic relevance are discussed by Sreekantaiah et al. (1994) and Gisselsson et al. (2001). Finally, cancer is a genetic disease, for acquired genetic aberrations cause the disease. Changes in antigen expression detected with immunohistochemistry in some instances reflects genetic alterations. The detection of these aberrations at the chromosome and gene levels improved diagnosis, prognosis, and therapy. Therefore, the combination of morphology with genetics is a major step toward a better understanding of human disease. A number of techniques that facilitate this combination are available, such as in situ hybridization, comparative genomic hybridization, expression profiling using array technologies, high throughput screening approaches, and phenotype/genotype correlations on the DNA, RNA, or protein level (Ried et al., 1999). Technological innovations such as image analysis systems, cytophotometric and integrated densitometric quantitation, and computer hard- and software development also assist in this effort.

GENETIC INSTABILITY OF TUMORS It is well established that cancer is caused by the accumulation of mutations in the genes that are directly responsible for cell birth or cell death. Genetic instabilities are a consequence of cancer mutations. One or more mutations initiate tumor growth, which give the tumorous cell a selective advantage over other cells. The clone derived from the tumorous cell then expands. Successive mutations occur, each followed by waves of clonal expansion. Information on the molecular and physiological bases of genetic instability of tumors is resulting in new approaches to treating cancer. One of two levels of genetic instability correlates with the vast majority of cancers. In most cancers genetic instability is observed at the chromosome level, resulting in losses

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or gains of whole chromosomes or large portions of them (Lengauer et al., 1998). On the other hand, in a small subset of tumors, genetic instability is observed at the nucleotide level and results in base substitutions, deletions, or insertions of a few nucleotides. An understanding of these instabilities is providing new insights into tumor pathogenesis. Four major types of genetic alterations that affect growth-controlling genes have been identified in neoplastic cells and are the basis of human cancers. 1. Sequence changes involving base substitutions, deletions, or insertions of a few nucleotides. This type of subtle change is exemplified by missense mutations in the K-ras gene, which occur in more than 80% of pancreatic cancers (Almoguera et al., 1988). These changes cannot be detected with cytogenetic analysis. 2. Alterations in chromosome number involve losses or gains of whole chromosomes and are found in almost all major types of human tumors. Losses of heterozygosity (losses of a maternal or paternal allele) are widespread. The average cancer of the colon, breast, pancreas, or prostate may lose ~25% of its alleles, and some tumors may lose more than half of their alleles (e.g., Vogelstein et al., 1989). Such cancers exhibit a true chromosomal instability that persists throughout the lifetime of the tumor. It is known that chromosome 10 is lost in glioblastomas, inactivating the tumor suppressor gene PTEN (Wang et al., 1997). The gain of chromosome 7 in papillary renal carcinomas indicates a duplication of a mutant MET oncogene (Zhuang et al., 1998). 3. Chromosome translocations are common in certain human cancers. Translocations such as fusions of different chromosomes or of normally noncontiguous segments of a single chromosome can be detected cytogenetically. At the molecular level, such translocations can produce fusions between two different genes, imparting to the fused transcript the tumorigenic properties. For example, in chronic myelogenous leukemias the carboxy terminus of the c-abl gene on chromosome 9 is joined to the amino terminus of the BCR gene on chromosome 22 (Nowell, 1997). Translocation can also cause gains or losses of chromosomal material and generate new gene products. Simple translocations are characterized by distinctive rearrangements of chromosomal segments in specific neoplastic diseases, including leukemias and lymphomas. These specific translocations are necessary for the development and progression of the neoplasms in which they occur. 4. Gene amplification is an important process in human cancers, as it is associated with tumor progression, has prognostic significance, and provides a target for therapeutics (Lengauer et al., 1998); an example is the amplification of HER-2/neu in breast cancers. At the cytogenetic level, gene amplifications can be detected as homogeneously stained regions or double minutes. At the molecular level, multiple copies of an amplicon containing a growth-promoting gene can be detected. The amplification of N-myc oncogene that occurs in about one-third of advanced neuroblastomas is a good example of tumor progression (Seeger et al., 1985).

Almost all solid tumors are genetically unstable. Translocations and gene amplifications add to the chromosomal abnormalities and may reflect additional mechanisms for generating genetic instability that occurs as tumors grow. Genetic instability is the cause of both tumor progression and tumor heterogeneity. As a result, no two tumors are exactly alike and no single tumor is constituted of genetically identical cells. Tumor

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heterogeneity is an obstacle in standardizing the diagnosis, as well as in selecting therapeutic strategies. However, it should be noted that although genetic instability is essential for neoplasia to develop, the instability may provide equally valid therapeutic targets.

TUMOR HETEROGENEITY Heterogeneity has been reported in a variety of human tumors. Intraindividual heterogeneity is defined as subpopulations of tumor cells found within one tumor. Heterogeneity among different tumors is called interindividual heterogeneity. The detection of heterogeneity at the tumor level is relevant to explanations of clonality differences, metastatic potential of human tumors, and response to therapy under different treatment regimes. The existence of heterogeneity can be explained by genetic instability of malignant progenitor or stem cells. In addition to other influences, heterogeneity might be one individual factor that explains differences in response and outcome of patients under treatment. Heterogeneity at the tumor cell level can be detected by histological, immunohistochemical, molecular genetic and flow cytometric methods, and polymerase chain reaction. Recently, multiparameter flow cytometry was used for detecting tumor cell heterogeneity (Könemann et al., 2000). This immunophenotyping, with its advantages of characterizing simultaneously a variety of different antigens, allows detection of interindividual as well as intraindividual heterogeneity and malignant subpopulations. The method provides the possibility of characterizing solid tumors according to their immunophenotype and DNA content. Molecules that are potentially involved in tumor invasion, metastasis, differentiation/maturation, and cell interactions can be chosen as target antigens. These include adhesion molecules, cell activation antigens, and cytokine and growth factor receptors.

Histological Microdissection Tissue heterogeneity of histological specimens is well known. Not only neoplastic cells are heterogenous; a tumor may contain a variable admixture of stromal cells, inflammatory infiltrates, endothelial cells, and preexisting tissue. This complexity hinders the study of molecular genetic alterations. Such studies require precise correlation of molecular genetic characteristics to well-defined cell populations. The presence of multiple cell types close to one another in the tumor may limit the precise significance of changes in specific cells. As a result, even sophisticated techniques become less useful when applied to bulk tissue. Study of uniform cell populations is a prerequisite to understanding differential gene expression in tumors. A number of mechanical techniques for microdissection have been developed to isolate cells for analysis from histological sections (Turbett et al., 1996; Youngson et al., 1995; Going and Lamb, 1996; Moskaluk and Kern, 1997; Lee et al., 1988; Zhuang et al., 1995). The most sophisticated technique is laser-assisted and suitable for microdissection of single cells with minimal risk of contamination (Becker et al., 1997). Some of the microdissection methods are satisfactory but also have certain disadvantages; for example, laser-assisted techniques require expensive equipment. To overcome some of the limitations of microdissection methods presently in use, recently a mechanical technique was developed by Harsch et al. (2001). This device

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consists of an ultrasonically oscillating needle and a piezo-driven micropipette for rapid and precise histological microdissection. The oscillating needle is used to fragment the tissue into subcellular particles that are aspirated into the pipette tip. The method can be applied to paraffin sections or unfixed cryostat sections. It allows a sharp demarcation between the dissected area and unwanted tissue that remains intact for further analysis. Individual colonic crypts can be dissected without collecting any adjacent stroma. Thus, the technique is useful for determining gene expression in defined cell populations.

ANTITUMOR VACCINES Advances in the molecular characterization of human tumors have led to a better understanding of tumor immunology. These advances reveal that cancer cells exhibit specific patterns of gene expression or molecular alterations, compared with normal cellular counterparts, resulting in the production of tumor-associated antigens. A number of such antigens are self-antigens, which allow conceiving and designing of specific vaccines against virtually every solid tumor. Thus, efficient cancer vaccines should be able to neutralize immune tolerance against such antigens. The idea of controlling cancer by stimulating the immune system is not a recent one. In fact, more than a century ago, bacterial extracts were used for stimulating tumor-specific immune responses (Coley, 1893). Subsequently, immunostimulatory cytokines were used against a number of cancers (Marincola et al., 1995). Passive transfer of cytotoxic immune cells (e.g., lymphocytes) was also tested in humans (Yee et al., 1997). Recently, a number of studies indicate that therapeutic vaccines might be useful in restoring immune defenses against cancer. The following discussion summarizes possible uses of cancer vaccines. An important future strategy for cancer immunotherapy is the use of the next generation of antigen-specific cancer vaccines. Until now, most clinical trials have been performed with end-stage cancer patients because data on vaccine-induced immune responses are limited. There are two variations of the development of antitumor vaccination strategies: (1) developing vaccines utilizing whole tumor cells and (2) working on vaccines targeting defined antigens. The advantage of tumor cell–based vaccines is that these in principle comprise all relevant tumor antigens. Consequently, there is no need for prior identification of the tumor antigens to be included in the vaccine. The limitation of this approach is that it is very difficult to understand the therapeutic effect of these vaccines on the disease. In contrast, the use of vaccines comprising defined antigens enables the improvement of vaccine strategies based on empirical findings (Offringa et al., 2000). This approach allows the systematic analysis of vaccine-induced immunity in relation to clinical response. This advantage strongly argues for the usefulness of antigen-specific anticancer vaccines. The protective effect of tumor cell vaccination is thought to involve defined T cell responses. However, only in selected cases does the detection of T cell immunity against defined antigens coincide with clinical response (Sun et al., 1999). For example, vaccination of patients exhibiting residual B cell lymphoma, using the tumor-specific immunoglobulin idiotype as an antigen, was shown to result in sustained molecular remission, accompanied by idiotype-specific T cell and antibody responses (Reichardt et al, 1999). Another positive example demonstrates that a human papillomavirus-specific vaccine can have therapeutic efficacy against benign neoplastic tumors such as genital warts (Lacey et al., 1999).

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Presently, vaccines expressing tumor-associated antigens are being tested in a therapeutic setting. A specific example of a candidate vaccine is Gastrimmune, which is being tested in patients with advanced gastric and pancreatic cancers (Greten and Jaffe, 1999). The aim of such testing is to specifically evoke or restimulate a specific antitumor immune effector response. The likely immune effector mechanisms involved in tumor control/elimination consist of innate immunity, cytotoxic T cells, NK cells, and antibodies. In addition, the activation of specific T helper cells is thought to be important not only to orient and regulate the immune response but also to sustain immune effector mechanisms in vivo (Bonnet et al., 2001). One way to use vaccines against cancer is by preventing infection by pathogens known to predispose to certain cancers. Approximately 16% of the worldwide incidence of cancer can be attributed to infectious pathogens (Ames et al., 1995). The objective is to decrease cancer incidence by using vaccines against the pathogens. A well-known recent example of this approach is the nationwide hepatitis B vaccination program in Taiwan. This resulted in the substantial decline in the incidence of hepatocellular carcinoma in children (Chang et al., 1997). Other cancers caused or facilitated by viruses against which experimental vaccines are available include Burkitt lymphoma, nasopharynx cancer, adult T cell leukemia, cervical carcinoma, B cell gastric lymphoma, and gastric carcinoma. The efficacy of DNA vaccines has also been compared with that of protein subunit vaccines. The use of plasmid DNAs as vaccines has several potential advantages in addition to ease of manipulation and preparation. For example, unlike most protein subunit vaccines, DNA vaccines are potentially able to stimulate both cell-mediated and humoral immunity. On the other hand, both subunit vaccines and DNA vaccines have perceived safety advantages over the use of live virus vaccines. Recently, Nass et al. (2001) have shown that a DNA vaccine expressing a single herpes simplex virus glycoprotein is safer than live virus immunization in immunocompromised animals and that the magnitude of protection in immuncompetent animals against subsequent challenge approaches the strength of protection achieved by sublethal infection. Another recent example of the development of a DNA vaccine is against the HER-2/neu expressing carcinomas. Foy et al. (2001) have utilized an in vivo murine tumor expressing human HER-2/neu for evaluating potential HER-2/neu vaccines consisting of full-length or various subunits of HER-2/neu delivered in protein or plasmid DNA form. This study demonstrates that protective immunity against HER-2/neu–expressing tumor challenge can be achieved by these vaccines. Partial protective immunity is also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of HER-2/neu. The mechanism of protection elicited by plasmid DNA vaccination is thought to be exclusively CD4-dependent, whereas the protection with ICD protein vaccination requires both CD4 and CD8 T cells. These early studies indicate that multiple forms of HER-2/neu vaccines would be more effective in eliciting the protective HER-2/neu–specific antitumor responses. It is expected that in the near future antigen-specific vaccines will be applied effectively to induce strong T cell immune responses in patients displaying less progressed stages of disease. A comprehensive discussion on the development of therapeutic cancer vaccines (molecular vaccines) has been presented by Moingeon (2001) and MonzaviKarbassi and Kieber-Emmons (2001).

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MOLECULAR GENETICS Basic mechanisms of pathogenesis can be elucidated through molecular genetic research. Based on such information, specificity, sensitivity, and efficiency of diagnostic and prognostic tests for many diseases can be improved. As a result, new insights into therapeutic approaches can be developed, and their effectiveness can be assessed more reliably. The Human Genome Project has focused attention on the association of mutations in nuclear DNA with human diseases. A complete working draft of the human DNA sequence was completed in spring 2000. This project continues to define disease-associated mutations, and the number of clinically useful molecular pathological techniques and assays has also been increasing. The technology is available to extract and amplify DNA from minuscule archival and fresh samples as diverse as blood, urine, sputum, and solid tissues, including fixed and paraffin-embedded tissues. Also, DNA can be purified even from dried blood spots for amplification and mutation analysis (Kiechle, 1999), thus permitting the study of an individual’s inherited genes and mutations. Hereditary information can also be obtained by assessing RNA, proteins, and enzyme activities. Such assessments can be morphological, immunological, or biochemical. The following examples indicate the usefulness of molecular pathology in better understanding diseases. The usefulness of identifying molecular alterations underlying neoplasia is obvious in borderline tumors. Whether such tumors should be classified as benign or malignant, and whether they represent a precursor of frank malignancy, is a matter of controversy despite extensive clinical and pathological studies. In many cases this problem can be solved by using the biology of tumors as the genetic indicator of malignancy. This approach is exemplified by mutations in the p53 tumor suppressor gene and Ki-ras oncogene, which are the most common genetic alterations in human cancers. These mutations are used as genetic indicators of malignancy. Methods are available to analyze abnormalities in these genes using paraffin sections of neoplasms (Frank et al., 1994; Caduff et al., 1995). Mutations in codon 12 of Ki-ras can be identified in DNA extracted from paraffin sections using an amplified created restriction site method, followed by confirmation using gene sequencing (Lin et al., 1993). Missense mutations in the p53 gene can be identified with an immunohistochemical surrogate to detect the nuclear accumulation of p53 protein that results from such mutations (Kerns et al., 1992). Recently, Caduff et al. (1999) have evaluated abnormalities in p53 and Ki-ras in malignant and borderline ovarian tumors of various histological types in paraffin-embedded tissues. The patterns of these genetic alterations in borderline and malignant neoplasms were compared and correlated with cell type and stage. This preliminary molecular analysis suggests that serous borderline tumors have the same molecular features usually associated with malignancy but are unlikely to represent a precursor of invasive serous carcinoma. On the other hand, mucinous borderline tumors may represent a precursor or variant of mucinous carcinoma of the ovary. Another example is the study of microsatellites that are short DNA loci having simple sequence repeats that are widely distributed throughout the human genome. They are a valuable source for human genetic linkage analysis and molecular cancer research

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because of allelic polymorphisms. In hereditary nonpolyposis colorectal cancer, an autosomal-dominant disorder accounting for 2–10% of all colorectal cancers, length alterations in single mononucleotide or dinucleotide repeats (microsatellite instability [MSI]) occur (Raedle et al., 1999). The MSI is used as a diagnostic criterion of replication errors caused by various mutations in at least five mismatch repair genes (Lynch and Smyrk, 1996). Therefore, MSI analysis is useful in clinical practice to identify patients with hereditary nonpolyposis colorectal cancer. Raedle et al. (1999) have presented a rapid DNA extraction method (rapid microsatellite analysis) for analyzing replication errors in paraffin-embedded tissues. Southern blotting and polymerase chain reaction are being used for detecting B- and T-cell clonality in lymphoproliferative diseases, including mantle cell lymphoma and lymphoma of the breast (Medeiros and Carr, 1999). Molecular genetic tests are currently important ancillary tools for the diagnosis and classification of malignancy, and their role is likely to increase in the future. The positive aspects of molecular genetics and molecular pathology mentioned above need to be balanced with ethical concerns to safeguard the rights and welfare of human subjects (Sobel, 1999). The state and federal regulations protecting patient’s privacy and welfare must be observed.

cDNA MICROARRAY TECHNOLOGY In conjunction with detailed understanding of the human genome, sophisticated methods are required for gene expression analysis and gene discovery. These approaches will provide insights into growth, development, differentiation, homeostasis, aging, and disease onset. One such recently introduced method is cDNA microarray or DNA-chip technology, which facilitates monitoring the expression of hundreds and thousands of genes simultaneously and provides a format for identifying genes as well as alterations in their activity (Kononen et al., 1998). Because of the wide spectrum of genes and endogenous mediators involved, this technology is helpful in recognizing chronic diseases. As the cDNA microarray technique allows large-scale expression analysis, it is well suited to observe the broad effects of oncogenic transcription factors on gene expression and potentially clarify their role in oncogenesis. The cDNA technology uses cDNA sequences or cDNA inserts of a library for polymerase chain reaction (PCR) amplification, which are arrayed on a glass slide with highspeed robotics at a density of 1,000 cDNA sequences per square centimeter. In other words, microarrays can be constructed from specific cDNA clones of interest, a cDNA library, or a selected number of open reading frames from a genome sequencing database to allow a large-scale functional analysis of expressed sequences. These microarrays serve as gene targets for hybridization to cDNA probes prepared from RNA samples of cells and tissues. A two-color fluorescence labeling technique can be used to prepare the cDNA probes so that a simultaneous hybridization, but separate detection, of signals provides the comparative analysis and the relative abundance of specific genes expressed. The cDNA technology is essentially an array-based, high-throughput protocol that determines gene expression and copy number survey of very large numbers of tumors. As many as 1,000 cylindrical tissue biopsies from individual tumors can be distributed in a single tissue microarray. Sections of the microarray also provide targets for parallel in situ

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detection of DNA, RNA, and protein targets in each specimen on the array. Moreover, consecutive sections allow rapid analysis of hundreds of molecular markers in the same set of specimens. Another advantage is that sufficient cDNA for hybridization to a microarray can be produced from as little as 1 mg of tissue. This technology can be used to profile complex diseases and discover novel diseaserelated genes. It can dissect complex human diseases by analyzing the pattern of gene expression. The cDNA microarray method could provide new targets for drug development and disease therapies and thus facilitate improved treatment of chronic diseases that are challenging because of their complexity. Listed below are examples of diseases whose molecular characteristics have been determined using gene arrays. Ljubimova et al. (2001) have used 11,000 gene microarrays for identifying gene expression profiles in brain tumors, including high-grade gliomas (glioblastoma multiforme [GBM] and anaplastic astrocytoma), low-grade astrocytomas, and benign extraaxial brain tumors (meningioma), and then compared them with normal brain tissue. In this study the gene array method was combined with reverse transcriptase (RT)-PCR and immunohistochemical evaluation of glial tumors. All GBMs overexpressed 14 known genes, whereas these genes were barely detectable in normal human brain tissue. This study also showed that laminin-80–containing GBMs recurred significantly sooner after surgical removal than did GBMs with a predominant expression of laminin-9. Thus, overexpression of laminin-8 in tumor blood vessel walls may be an indicator of time to recurrence for patients with GBM. Another example of the involvement of many different genes in a cancer is renal cell carcinoma. This cancer is one of the 10 most frequent malignancies in western countries. Genes involved in the initiation and progression of this cancer include the von Hippel–Lindau gene on chromosome 3p, the epidermal growth factor receptor gene on chromosome 7p, the transforming growth factor gene on chromosome 2p, and the c-myc oncogene on chromosome 8q (Siezinger et al., 1988; Moch et al., 1998; Lager et al., 1994; Yao et al., 1998). Other genes involved in renal cancer are currently not known. Moch et al. (1999) have combined tumor arrays and cDNA arrays for rapid identification of genes and their role in renal cell carcinoma. They constructed a kidney cancer tissue array consisting of 532 renal tumors, 386 of which had clinical follow-up data available. There were 89 differentially expressed genes in the cancer cell line CRL-1933, one of them encoding for vimentin. Vimentin expression was significantly associated with poor patient prognosis independent of grade or stage. cDNA microarray technology has also been used for verifying the involvement of a number of genes in another complex disease, rheumatoid arthritis (Heller et al., 1997). In this disease inflammation of the joint is caused by the gene products of many different cell types present in the synovium and cartilage tissues plus those infiltrating from the circulating blood. In this study the presence of gene products, such as matrix degrading metalloproteinase (MMP), macrophage inflammatory protein (MIP), and human matrix metalloelastase (HME), was verified. The expression profiles of the genes demonstrate the utility of the microarrays in determining the hierarachy of signaling events. The downstream effects of both PAX3 and PAX3-FKHR on NIH 3T3 cells with cDNA microarrays has also been monitored (Khan et al., 1999). This study elucidated the pattern of gene expression induced by these two oncogenic transcription factors in these cells; these factors showed significant myogenic properties. Other recent examples of the application of gene arrays for determining the molecular parameters of individual tumors

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are ovarian and cervical cancers and metastatic versus primary breast cancer (Ono et al., 2000; Shim et al., 1998; Nacht et al., 1999). New subclasses of leukemia have also been identified using gene arrays, which have become critical for the successful treatment of patients (Golub et al., 1999). Because the individual arrayed tissue samples are very small (0.6 mm in diameter), one might ask if these specimens are representative of their donor tumors! To answer this question, Nocito et al. (2001) studied a set of 2,317 bladder tumors that had been previously analyzed for histological grade and Ki-67 labeling index. The histological grade and the Ki-67 labeling index were determined for every arrayed tumor sample. The grade and Ki-67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections. On the basis of this evidence, it can be stated that intratumor heterogeneity does not significantly affect the ability to detect clinicopathological correlations on the tissue microarrays. It is concluded that tissue microarray is an important tool for rapid identification of biological or clinically significant molecular alterations in tumors.

ANGIOGENESIS Two distinct processes, vasculogenesis and angiogenesis, form blood vessels. Vasculogenesis is responsible for the de novo differentiation of endothelial cells from mesodermal precursors and occurs during embryonic development, leading to the formation of a primary vascular plexus. Angiogenesis, on the other hand, is the process of sprouting and configuring new blood vessels from preexisting ones. It is a complex phenomenon comprising a series of cellular events that lead to the neovascularization associated with the process of tumor growth, metastasis, inflammation, and wound healing (Fig. 1.5/Plate 1A). Angiogenesis that occurs in wound repair and formation of collateral blood vessels following an infarct, ischemia, or reduced blood flow is advantageous for

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normal tissue function. It should be noted that tumor angiogenesis is not sufficient to cause tumor spread and patient death. Tumor cells must also proliferate, penetrate host tissues and vessels, survive within the vasculature, escape the host immune system, and then begin growth at a new body site (Weidner, 1998). Before discussing the role of angiogenesis in disease, it is relevant to explain the process of angiogenesis. The complex process of angiogenesis includes the recruitment of nearby endothelial cells, their activation, degradation of the vascular basement membrane, proliferation and form a new capillary (Albini et al., 2000). Tumor-induced endothelial cell activation leads to the acquisition of a phenotype characterized by chemotactic motility, basement membrane invasion, and proliferation. These events are followed by differentiation into a new vessel. During the last decade there have been significant advances in the understanding of functional mechanisms of the molecules involved in angiogenesis. Angiogenesis is mediated by multiple positive and negative regulator molecules released by tumor cells, intratumoral macrophages, mast cells, and endothelial cells. The balance of the effects of these mediators determines the outcome of this process. At least three groups of extracellular signals are involved in angiogenesis: (1) soluble growth molecules such as acid and basic fibroblast growth factors and vascular endothelial growth factor (discussed later) that affect endothelial cell growth and differentiation; (2) factors such as transforming growth factor and angiogenin that inhibit proliferation and enhance differentiation of endothelial cells; (3) extracellular matrix–bound cytokines released by proteolysis, which contribute to angiogenic regulation. Other growth factors implicated in different steps of angiogenesis are platelet-derived growth factor, hepatocyte growth factor, and angiopoietins 1 and 2. Also, various endothelial surface molecules, such as CD31, CD144, and integrins, play a role in angiogenesis. Some of the above-mentioned secreted factors are angiogenic, whereas others are angiostatic. Thus, angiogenesis is mediated by multiple positive and negative regulatory molecules released by both tumor cells and the surrounding normal cells. The balance between these regulators determines whether or not neovascularization will occur. Indeed, antiangiogenic therapy is based on the use of negative regulators of neovascularization aimed at suppressing the proangiogenic signal or increasing the inhibitory signals. Albini et al. (2000) have used the gene therapy approach using class I interferons for effectively inhibiting tumor angiogenesis and growth of vascular tumors. Although overwhelming evidence indicates that endothelial cells are central to the angiogenic process, the following discussion proposes the role of tumors in the formation of blood vessels. According to Maniotis et al. (1999) and Folberg et al. (2000), blood vessels of malignant eye tumors known as uveal melanomas are formed by tumor cells instead of endothelial cells. These highly aggressive and metastatic cells are capable of forming in vivo and in vitro vascular channels, which consist of a basement membrane that stains positive with the periodic acid–Schiff (PAS) reagent in the absence of endothelial cells and fibroblasts. The generation of such channels is termed vasculogenic mimicry. This evidence suggests that angiogenesis may not be the only mechanism responsible for creating tumor microcirculation. Therefore, methods used to identify the tumor microcirculation by staining endothelial cells may not be applicable to tumors that express vasculogenic mimicry. However, certain aspects of the concept of vasculogenic mimicry have been questioned by McDonald et al. (2000). They indicate that PAS-stained channels do not

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represent the microvascular architecture, and endothelial cell–lined blood vessels are also present in uveal melanomas. They moreover report that tumor cell-lined vessels are infrequent in these melanomas. Nevertheless, tumor cells can acquire a new phenotype and participate in the formation of blood vessels. It is concluded that the extent and the pathophysiological significance of cancer cells becoming lining cells and participating in the formation of blood vessels in tumor is still unclear. Information on the angiogenesis regulatory molecules has produced new therapeutic strategies for suppressing angiogenesis and tumor growth or promoting angiogenesis against coronary and peripheral ischemia and stimulation of wound healing (Thompson et al., 1999; Kahn et al., 2000). Limited space does not allow discussion of these aspects of angiogenesis, except for VEGF, which is the most potent angiogenic factor (see pages 23–24). Angiogenesis plays an important role in the development and progression of a number of disease states, including various cancers, diabetic retinopathy, macular degeneration, psoriasis, and rheumatic arthritis. The tumor microcirculation plays a key role in hematogenous dissemination of cancers. There is compelling evidence that angiogenesis is indeed critical for tumor growth progression and metastasis because tumors require new blood vessels to achieve a size larger than 2–3 mm. Also, a considerable amount of evidence suggests that tumor angiogenesis is crucial for the growth of solid tumors in vivo (Folkman, 1996). The growth of tumors (including solid tumors) must be preceded by an increase in capillaries and newly formed blood vessels that provide tumor cells with oxygen and nutrients as well as paracrine mediators. The blood vessels also remove waste products. A large number of tumor blood vessels increases the opportunity of the tumor cells to enter the circulation. In fact, the newly formed capillaries usually have a fragmented basement membrane, facilitating easier invasion. In the prevascular phase, with little or no angiogenic activity, the tumor is unable to expand beyond a few cubic millimeters, but once angiogenic factors are released in sufficient number, the onset of angiogenic activity stimulates rapid expansion of the tumor. The microvessel density of the tumor mass, a measure of tumor angiogenesis, correlates with metastasis and can be used as an independent prognostic factor in the management of cancer (Jacquemier et al., 1998). A number of studies indicate that microvessel density gives prognostic information on breast cancer (Weidner et al., 1993). With respect to prognostic carcinoma, it is thought that a low vascular density correlates with significantly longer survival duration than with carcinomas having high vascular density. Thus, neovascularization has proven to be an independent predictor of pathological state in prostatic carcinoma. A correlation between the endocrine differentiation and increased neovascularization in prostatic cancer has also been reported (Grobholz et al., 2000). High-grade tumors with a high neuroendocrine differentiation and increased neovascularization indicate high risk and unfavorable outcome. Although the role of angiogenesis as a prognostic factor has been most widely analyzed in breast cancer, angiogenesis also plays an important prognostic role in other carcinomas such as gastric cancer. This cancer is a highly aggressive malignancy with poor prognosis and low survival rates. Sanz-Ortega et al. (2000) have evaluated advanced gastric cancers for the expression of oncogenes HER-2/neu (c-erbB-2), c-myc, and epidermal growth factor receptor, as well as microvessel density. Avidin-biotin immunohistochemistry using CD34 stained paraffin sections has shown that tumor angiogenesis is the most important independent prognostic indicator to predict overall survival.

Introduction

23

Immunohistochemistry using primary antibody against rabbit antihuman VEGF (diluted 1:1000) has demonstrated that angiogenesis is also a vital process in cartilaginous tumors and that VEGF expression by malignant chondrocytes is required for the formation of intracartilage vessels (Ayala et al., 2000). Intracartilage vessels might be involved in the acquisition of metastatic potential by cartilage tumors. Squamous cell carcinoma is also characterized by a richly vascularized stroma and overexpression of VEGF. This carcinoma of the skin is a malignant tumor of epidermal keratinocytes with a destructive growth pattern, and it has the ability to metastasize. It has been demonstrated that selective overexpression of VEGF in highly differentiated squamous cell carcinomas is sufficient to induce tumor invasiveness as well as to promote tumor growth and angiogenesis (Detmar et al., 2000). The tumor stroma also plays an active role in the progression of this cancer. As stated earlier, angiogenesis plays a role in repairing blood vessel injury. Two systems, angiogenesis and hemostasis, remain poised for repair of blood vessel injury. At the site of blood vessel injury, adhered platelets secrete both positive and negative regulators of angiogenesis, mainly from internal The positive regulators include VEGF; negative regulators include platelet factor 4. Hepatocyte growth factor affects both stimulation and suppression of angiogenesis. On the other hand, the hemostatic system maintains the liquid flow of blood by regulating platelet adherence and fibrin deposition. Browder et al. (2000) have discussed in detail how angiogenesis is coordinated by and with hemostasis during blood vessel repair. In conclusion, sufficient evidence is available indicating that assessment of microvessel density is very useful in tumor biology. However, consensus on the prognostic value of angiogenesis is lacking. The main reasons for conflicting results consist of the study of different angiogenic-regulating factors, the use of varying methodologies for measuring microvessel density, and the significant intraobserver variation that exists in interpretation of the number of positive vessels and the optimal way in which fields are selected, that is, hot spot (the most vascular area of the tumor) versus general counting of vessels. These reasons do not allow meaningful comparison among the results reported in various studies. Standardization of processing conditions, such as tissue section preparation, staining, careful selection of the hot spot, and a strict protocol for defining microvessels, can achieve adequate reproducibility. However, despite these precautions, manual counting of microvessels and selection of the hot spot are still subjective and therefore not always fully reproducible. These problems can be significantly minimized by using fully automated microvessel counting and hot spot selection by image processing of whole tumor sections, for example, in invasive breast cancer (Beliën et al., 1999). In comparison with the manual method, the automated procedure reduces the microvessel measurement time when the complete tumor is scanned, achieves greater accuracy and objectivity of hot spot selection, and allows visual inspection and relocation of each measurement field awards. The reasons for contradictory interpretations and potential remedies have been presented in more detail by Hansen et al. (1998).

Vascular Endothelial Growth Factor The vascular endothelial growth factor (VEGF) is a member of the six-member VEGF family: VEGF, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. These

24

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members have overlapping but specific roles in the growth of new blood vessels. The following discussion is limited to only one member, VEGF (also known as vascular permeability factor), which is the most important and most frequently studied angiogenic factor. It is a homodimeric 34–42 kDa glycosylated heparin-binding glycoprotein. Alternative exon splicing of the VEGF gene produces multiple species of mRNA, which encode different VEGF protein isoforms having subunit polypeptides of 121, 145, 165, 189, or 206 amino acid residues (Neufeld et al., 1999). Vascular endothelial growth factor has three receptors [VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (FLT-4)], each consisting of seven immunoglobulin-homology domains, a transmembrane sequence, and an intracellular portion containing a split kinase domain (Shibuya, 1995). Ligand (VEGF) binding induces receptor dimerization and subsequent auto/transphosphorylation. The receptors have distinct roles in vasculogenesis and angiogenesis during embryonic development. Precise roles of the three receptors have been discussed by Veikkola and Alitalo (1999). Transcription of VEGF mRNA is induced by a variety of growth factors and cytokines, including platelet-derived growth factor-BB, epidermal growth factor, tumor necrosis transforming growth and (Ferrara and Davis-Smyth, 1997). Tissue oxygen tension tightly regulates VEGF levels, and exposure to hypoxia rapidly and reversibly induces VEGF expression through both increased transcription and stabilization of the mRNA (Levy et al., 1996). Hypoxic upregulation of VEGF thus provides a compensatory mechanism by which tissues can increase their oxygenation through induction of blood vessel growth. Normoxia downregulates VEGF production and leads to regression of certain newly formed blood vessels. By these opposing processes the vasculature becomes matched to the tissue oxygen demands (Veikkola and Alitalo, 1999). The vascular endothelial growth factor is produced by tumor cells, macrophages, and endothelial and smooth muscle cells. It induces vascular endothelial cell migration, enhances vascular permeability, and promotes extravasation of plasma proteins from tumor vessels to form an extracellular matrix, facilitating inward migration of endothelial cells (Callagy et al., 2000). These characteristics impart selectivity to VEGF for endothelial cells. The vascular endothelial growth factor is involved in angiogenesis in a wide variety of biological systems, including the female reproductive cycle, wound healing, and tissue repair. Proliferation of blood vessels during the formation of the corpus luteum in the ovary and during the growth of endometrial vessels in the uterus occurs upon expression of the VEGF mRNA and protein (Ferrara and Davis-Smyth, 1997). This factor is also detected during angiogenesis occurring at the site of embryo implantation in the uterus (Shweiki et al., 1993). In ischemic cardiac tissue, VEGF mRNA is increased, suggesting the involvement of this factor in the growth of collateral blood vessels (Hashimoto et al., 1994). In addition to its role in physiological angiogenesis, VEGF is active in pathological neovascularization. For example, squamous cell carcinoma of the skin strongly expresses VEGF (Weninger et al., 1996). In fact, tumoral VEGF correlates with prognosis in a variety of tumors, including breast cancer and malignant mesothelioma (Fig. 1.6).

Immunohistochemical Localization of Vascular Endothelial Growth Factor The following method is recommended for immunostaining vascular endothelial growth factor (VEGF) (Callagy et al., 2000). Breast cancer tissues (including the invasive

Introduction

25

edge of the tumor) are fixed with formalin and embedded in paraffin. Sections thick) are mounted onto adhesive-coated slides, dried, and then deparaffinized. The sections are treated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. After washing in PBS, the sections are placed in 10 mM sodium citrate buffer (pH 6.0) and boiled for 5 min in a microwave oven to unmask the antigens. They are washed in Tris buffer sodium chloride (25 mM Tris-HCl [pH 7.6] and 150 nM sodium chloride) and incubated in normal goat serum (diluted 1:10 with TBS) for 30 min to block nonspecific staining. The sections are incubated in the rabbit polyclonal anti-VEGF (Santa Biotechnology, CA) and diluted 1:100 for 30 min at room temperature. Antigen-antibody reaction is detected using the biotin-streptavidin–based detection kit (Dako). The reaction is developed using DAB+hydrogen peroxide and counterstained with Mayer’s hematoxylin. Exclusion of the primary antibody serves as a negative control. The results of this procedure are shown in Fig. 1.7. Telepathology (Telemedicine) Telepathology, introduced by Weinstein et al. (1987), is a pathology practice that requires telecommunication technologies to transmit digital images to distinct sites for diagnostic, consultation, and educational purposes. Telepathology is an affordable option in places where a pathologist is unaffordable, such as rural hospitals that are too small to support a pathologist. In addition, telepathology facilitates seeking a second opinion on a difficult case. It is thought to be accurate and cost-effective, and its advantages outweigh the problem of waiting longer to have a slide read. Also, the resolution obtainable with videomicroscopy is thought to be adequate and appropriate for diagnosis. Telepathology is expected to become an integral part of medical practice for practical, economic, and humane reasons.

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Telepathology can be divided into two major modalities: static imagery and dynamic (real-time) imagery. Each of these two methods has advantages and limitations. Static imagery (the store-and-forward method) involves capturing of still images from a microscope and transmitting them through a point-to-point connection or by transmission control protocol/internet protocol. The still digital images selected at the remote site are transmitted at a later time for remote diagnosis. The images provided are of superior quality, but the number of images is limited. It is usually not feasible to transmit the images in real time, and the selection of images by the remote site requires two pathologists to share the interaction. The images can be transmitted over the Internet because bandwidth (role of data transmission) requirements are low for the static imagery. In static imagery, expedient delivery of high-resolution images can be achieved by attaching pathology images with an electronic mail message. This method is applicable when real-time consultation is not required. Despite the low cost and simplicity of static imagery,

Introduction

27

it has certain shortcomings. Because static imagery uses relatively low-resolution digital photomicrography, it requires high optical magnification to allow adequate examination of diagnostic images. This results in the need to collect and transmit multiple image files. Furthermore, static image acquisition means that fields for imaging are preselected by a person other than the telepathology consultant, leading to unattended field selection biopsy error (Weinstein et al., 1997). However, high-resolution digital scanning cameras allow the acquisition of digital images up to 3,400 × 2,700 pixels of resolution. These images can be captured at a relatively low optical magnification and digitally magnified multiple times without visible degradation. They can be scrolled at different magnifications in computer, simulating light microscopy. This high-resolution digital photomicrography and the Internet have been used for telepathological gastrointestinal biopsy consultations (Singson et al., 1999). In contrast to static imagery, in dynamic imagery the consultant examines a histological or cytological slide from a remote site by using sophisticated robotic microscopes that transmit real-time digital images through fast and expensive telecommunication links that provide very high band widths. According to Weinstein (1996), low-resolution dynamic images are more useful to a pathologist than high-resolution static images. The diagnostic accuracies for static imaging and dynamic imaging are 88% and 96–97%, respectively. The latter range falls within the acceptable range for surgical pathology. Although the real-time telepathology shows a higher diagnostic accuracy, static imagery continues to be the dominant method used. Static imaging is adequate in those cases where tissue sampling is not a problem. Attempts have been made to develop systems that combine the advantages of static imagery and dynamic imagery. Such systems have been described by O’Brien et al. (1998). However, few of these systems have been implemented because of their complexity. Recently, a new hybrid telepathology system has been described, which achieves dynamic real-time microscopic video transmission for providing dynamic imaging. The implementation of this system is awaited. Imaging standards remain an issue in telepathology. Lack of critical literature in this field is also a barrier to further development and acceptance of this technology. Furthermore, standards must be developed and accepted for the types of cases that will be diagnosed and for protecting patients’ privacy. In addition, telepathology equipment is more expensive than teleradiology equipment. The most extensive and well-known telepathology service is part of the U.S. Armed Forces Institute of Pathology. This service offers the diagnostic evaluation of microscopic still images sent via e-mail (http://www.afip.org/). Recently, telemicroscopy via Internet browsers such as Netscape Navigator and Internet Explorer was introduced by Wolf et al. (1998a, b). They reported a new concept in Internet functionality by demonstrating how Internet browsers with Java support can use remote control of computer-controlled devices such as an automatic microscope. More recently, Petersen et al. (2000) reported how this technology can be used for image transfer and communication between pathologists or research scientists. Essentially, it is based on a conventional light microscope with a video camera, which in turn is connected to a computer with a frame grabber and Internet access. This Telemic system allows the user to show and discuss microscope images with any pathologist who is connected to the Internet. For inquiries about the software and information on the installation, the reader should contact the Telemic homepage at http://amba.charite.de/telemic

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FUTURE OF IMMUNOHISTOPATHOLOGY An increasing understanding of the molecular changes associated with various tumor groups, and the genetic variability within them, is beginning to provide important new information about clinical progression and prognosis (Graadt van Roggen et al., 1999). Many malignant tumors carry chromosomal aberrations detectable at a cytogenetic or molecular level. Some of these changes are nonrandom and associated with specific tumor types (Ladanyi, 1995). Chromosomal analysis aimed at detecting specific alterations is the most effective variable in resolving the frequent diagnostic dilemmas. The detection and description of apparent tumor-specific genetic alterations within the sarcoma group are already beginning to play an increasingly important role in unraveling and understanding the molecular biology of tumorigenesis. For example, mutations in the retinoblastoma gene (Rb) have been detected in a large proportion of high-grade sarcomas (Sreeckantaiah et al., 1994). Also, the coinactivation of p53 and Rb indicates that both genes may be involved in tumorigenesis of certain sarcomas. In fact, the identification of increasing numbers of tumor-specific genetic alterations has become an helpful adjunct to histopathological assessment in reaching a correct diagnosis. The above observations suggest that an accurate histological classification of tumor types is useful in establishing meaningful clinical trials of optimal management strategies. The relationship between immunohistopathology and surgical pathology becomes apparent when one considers that a large number of monoclonal antibodies is being produced that detect cells at each stage of cancer development. These antibodies are directed against antigens that determine levels of proliferation, angiogenesis, proteolysis, and cell adhesion (Elias, 1999). Thus, it has become possible to determine biochemical alterations occurring during the cell’s progression to malignancy. In addition, new oncogenes are being discovered at a rapid pace. These developments are helping pathologists and oncologists to refine therapeutic and prognostic decisions. Furthermore, these advancements, along with increasing understanding of the molecular and cellular mechanisms of cancer, are expected to lead us to the evaluation of a person’s risk of developing cancer. The ultimate goal, of course, is to prevent cancer. The relationship between gene expression profiles and cellular behavior in humans is largely unknown, and expression patterns of individual cell types have yet to be precisely measured (Emmert-Buck et al., 2000). Although we know that the human genome consists of 32,000 genes, at present the function of only a relatively small percentage of genes is known. However, it is hoped that our understanding of how gene expression modulates cellular phenotype and response to the environment will be achieved within the next few years or a few decades. In June 2000, the International Human Genome Project and Celera Genomics Corporation announced the completion of a “working draft” of the human genome sequence, the genetic code that carries the instructions allowing us to develop, grow, and live. It is possible now to understand the secrets of life processes to an extraordinary degree, to personalize medicine and offer clues to the differences and remarkable similarities among us. Human genome information in concert with full-length cDNA sequencing of all genes will also lead us to an exciting new paradigm in biomedical research known as molecular profiling (Emmert-Buck et al., 2000). Molecular profiling will facilitate the identification of individual genes and collection of genes that mediate particular aspects of

Introduction

29

cellular physiology and pathology, thus improving our understanding and treatment of diseases. I am confident that with the approach of the postgenome era, an ever-increasing number of human genes will be discovered and their functions elucidated. Combined with the knowledge of human gene polymorphism, genotyping will allow prediction of the genetic predisposition to certain diseases, such as cancer. The new millennium will usher us in a new era of disease-predictive medicine.

PREPARATION OF BUFFERS Tris-buffered saline (TBS) (Stock solution) Tris NaCl Distilled water Adjust pH to 7.8 Distilled water to make 5 liter

303 g 450 g 4 liter 185 ml HCl (32%)

Dilute 10 times with distilled water before use

Phosphate-buffered saline (PBS) (Stock solution) Disodium hydrogen phosphate dihydrate Potassium dihydrogen phosphate NaCl Distilled water Adjust pH to 7.4 Distilled water to make 5 liter

70.5 g 10.5 g 450 g 4 liter

Dilute 10 times with distilled water before use

Citrate buffer for heat-induced antigen retrieval Citric acid monohydrate 2.1 g Distilled water 4 liter Adjust pH to 6.0 with 13 ml 2 N NaOH solution

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Chapter 2

Antigens and Antibodies

It is instructive to define relative terminologies. An antigen is a molecule that combines with a specific antibody but which itself may not necessarily be immunogenic. An immunogen is a cell or macromolecule that stimulates a specific immune response. An epitope (an antigenic determinant) is the site on a complex antigenic molecule which is recognized by the antibody. An antibody (immunoglobulin) is a glycoprotein molecule produced by differentiated B lymphocytes when stimulated by an antigen. Immunoglobulin G (IgG) is the most abundant class of immunoglobulins in human serum. IgG is the primary Ig molecule produced during the secondary immune reaction to the antigen. Immunization is the administration of an antigen to an animal to evoke the production of antibodies. Serum is the blood plasma from which the fibrogen has been removed. Mammalian sera contain ~8% (w/v) protein, consisting of approximately equal proportions of albumin and globulin. Antiserum is the serum containing antibodies to an antigen. Fab is the fragment of an immunoglobulin (Ig) that binds to an antigen and is produced by treating the Ig molecule with the enzyme papain. is the fragment of an Ig molecule which contains both antigen-binding sites and the disulfide bridge. It is produced by treating the Ig molecule with the enzyme. Fc fragment is the part of an Ig molecule that has no antigen-binding activity but binds to FC receptors on phagocytes and may activate complement. A clone is a group of daughter cells that are produced from a single cell. Hybridoma is the cloned hybrid cells formed by the fusion of an antibody-forming cell and a malignant myeloma cell. A hybridoma grows continuously and produces antibodies of a single specificity termed monoclonal antibodies. Affinity is the association constant at the equilibrium between an epitope and a single antigen-binding site of the antibody, independent of the number of sites. This term describes the strength and the stability of the binding. Specificity refers to the selective binding between an antigen and its corresponding antibody. Titer is the measure of units of antibody per unit volume of serum. The concentration of the antibody is determined by titration.

ANTIGENS An antigen is a substance that reacts specifically with receptors on the surface of lymphocytes and with their soluble products such as antibodies. Antigens usually are large, complex protein or polysaccharide molecules with molecular weights usually greater than 31

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40,000. However, the molecular weight, for example, may vary from 15,000 (hen egg white lysozyme) to 2,000,000 (keyhole limpet hemocyanin) daltons. Protein antigens function as the most potent immunogens, and polysaccharide antigens rank second. For cell-mediated immunity, only proteins serve as immunogens. Certain nucleic acid types such as Z-DNA and other molecules can also stimulate antibody production. Antigens can be defined on the basis of four immunological properties: immunogenecity, antigenicity, allerogenicity, and tolerogenicity. The ability of a substance to induce an immune response is called immunogenicity. Most antigens have a variety of different antigenic determinants (epitopes) on their surfaces, which stimulate antibody production. Antigenicity is the ability of an immunogen to combine with an antibody or cell surface receptors. Allerogenicity is the ability to induce various types of allergic responses. Allergens are immunogens that tend to activate specific types of humoral or cell-mediated responses having allergic manifestations (Kuby, 1992). Tolerogenicity is the capacity to induce specific immunological nonresponsiveness in either the humoral or the cell-mediated systems. In other words, experimentally induced tolerance can be defined as a state in which an animal fails to respond to an antigen that would normally be immunogenic. It is not known whether similar mechanisms generate both naturally acquired self-tolerance and experimentally induced tolerance. Immunogens induce an immune response only if they are recognized as foreign (nonself). A case in point is protein bovine serum albumin, which is immunogenic in sheep but not in cows. Most large antigens have multiple reactive sites, or epitopes, on their surfaces, which can induce production of specific antibodies. Antibodies do not recognize the whole immunogen but only small regions (epitopes). Each type of antibody binds to its own inducing epitope. For example, lysozyme, an enzyme that degrades the carbohydrate coat of bacteria, induces several different antibodies, each of which binds to a particular epitope on the lysozyme molecule (Lodish et al., 2000). Although different epitopes on lysozyme differ greatly in their chemical properties, the interaction between lysozyme and antibody is complementary in all cases. In other words, the surface of the antibody’s antigenbinding site fits into that of the corresponding epitope as if they are molded together. The intimate contact between these two surfaces, stabilized by numerous noncovalent bonds, is responsible for the exquisite binding specificity shown by an antibody.

Epitopes Immune cells do not interact with or recognize an entire immunogen molecule; instead, lymphocytes recognize discrete sites on the antigen called epitopes (antigenic determinants). Epitopes are the immunologically active regions of an immunogen which bind to specific membrane receptors for antigen on lymphocytes or to secreted antibodies. Interaction between lymphocytes and a complex antigen may involve several levels of antigen structure. In the case of protein antigens, the structure of an epitope may involve elements of the primary, secondary, tertiary, and even quaternary structure of the protein. In the case of polysaccharide antigens, extensive side-chain branching via glycosidic bonds affects the overall three-dimensional conformation of individual epitopes. Epitopes are small linear sequences of amino acid residues, branched sequences of carbohydrate, or “shape” sequences brought about by the folding of a protein molecule. The

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33

remaining part of the protein antigen molecule is the carrier for the epitope. An antigen such as bovine serum albumin has several different epitopes on its surface, each of which stimulates the cell having the appropriate receptor. Because an epitope usually comprises a sequence of approximately three to eight amino acid residues, antibodies should be regarded as site- or region-specific detection molecules instead of antigen-specific molecules.

ANTIBODIES Immunohistochemical labeling with antibodies has become the most sensitive and powerful method for localizing antigens in situ and thus for characterizing cells and their components and their functions. In this context the importance of antibody specificity and selectivity for the antigen cannot be overemphasized. The specificity relies entirely on the properties of the primary antibody, independent of the procedure used for detection. Although both monoclonal and polyclonal primary antibodies can be generated or purchased, the former are preferred and are in much wider use because of their far greater specificity. The basic structure of an antibody molecule is Y-shaped, with the two tips designed to recognize and bind antigens (Fig. 2.1). The tips, through the disulfide bridge, are free to bend with respect to each other. This property increases the binding strength of the antibody for antigens that have multiple, adjacent antigenic determinants and for antigens that are closely packed together. The remainder of the antibody molecule enables it to interact with other proteins, preventing undesirable company. Antibodies are molecules secreted by terminally differentiated B cells (a type of lymphocyte) known as plasma cells. Nearly all rabbit primary antibodies and most mouse monoclonal antibodies are immunoglobulins (Igs). There are five classes of Igs that differ structurally and functionally. Immunoglobulin G (IgG) molecules are the major class of Igs in the blood, which are predominantly produced in the secondary immune response. Monoclonal antibodies have been termed magic bullets and hailed in publications as the cure for cancer. Belief in this idea was strengthened by the successful clinical results of mouse

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anti-idiotypic monoclonal antibodies in the treatment of lymphomas and leukemias (Levy and Miller, 1983) and by FDA approval in 1986 of the OKT3 anti-CD3 mouse monoclonal antibody for acute renal transplant rejection (Shield et al., 1996). However, this excessive optimism has been questioned because of adverse clinical and laboratory findings. For example, when rodent monoclonal antibodies were used therapeutically, a human antimurine antibody response developed in up to 50% of treated patients (Khazaeli et al., 1994). Effector functions of mouse antibodies also have proven less efficient in the human context (Gavilondo and Larrick, 2000). The biological half-life of these antibodies is shorter than that of human immunoglobulins. This characteristic of mouse antibodies limits their usefulness. These limitations can theoretically be overcome by using monoclonal antibodies of human origin (Thompson, 1988); however, human monoclonal antibodies from hybridomas and lymphocyte cell lines are very difficult to generate. Nevertheless, beginning in 1994, the FDA approved a number of antibodies to combat human diseases, including follicular non-Hodgkin’s B cell lymphoma, breast cancer, and rheumatic arthritis (Grillo-Lopez et al., 1999; Weiner, 1999; Maini et al., 1999).

Polyclonal Antibodies Both polyclonal and monoclonal antibodies have advantages and limitations with regard to their generation, specificity, cost, and overall applications. Polyclonal antibodies possess higher affinity and wider reactivity but lower specificity. They have the advantage of detecting many types of epitopes and recognizing antigens of different orientations. Polyclonal antibodies show greater stability at varying pH levels and salt concentrations and are more useful for preadsorption controls. They are simpler to produce in a shorter duration, and there is no risk of loss of clones. In addition, large animals (e.g., rabbits and horses) can be used to recover large volumes of antibody-rich serum. However, a fresh batch of the serum is required when the original stock is exhausted. This replacement results in batch-to-batch variation, which may result in differences in antibody reactivity and titer (Nelson et al., 2000). Such differences result in a lack of reproducibility. Polyclonal antibodies are composed of multiple species of immunoglobulins directed toward several epitopes within a particular antigenic molecule. Moreover, only a minor proportion of the antibody present in the polyclonal antiserum is specific for the immunizing antigen. The remainder may consist either of antibodies produced by the animal in the past in response to previous antigenic stimuli or of antibodies against contaminating antigens present in the immunizing preparation (Mason et al., 1983). Even the antibodies in the polyclonal antiserum that are specific for the immunizing antigen are usually heterogeneous and are directed against a number of different epitopes on the immunizing antigen. However, although whole sera or whole IgG fractions of polyclonal antibodies often have problems, affinity purification against an antigen affinity column can dramatically improve the usefulness of the polyclonal reagents. Thus, a mixture of isoforms of antibodies to different epitopes is obtained. These epitopes are still relatively unique to the antigen involved. The main advantage is that 100% of the antibody in these preparations reacts with the antigen, often at multiple and therefore additive sites. This approach is analogous to mixing monoclonal antibodies to label different epitopes together. Although such reagents

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35

are not commonly commercially available, their usefulness in immunohistochemistry should not be minimized. The procedure to produce such reagents requires the ability to prepare significant amounts of the pure antigen, either a purified protein (or carbohydrate) or even a complex peptide. The antigen is covalently linked to the beads of a column (usually cyanogen bromide–activated Sepharose). The antibody preparation is passed over the column and the antibody reactive with the antigen sticks. The excess (usually 99% of the total IgG) (flow through) is then washed away from the column, and the specific antibody is eluted from the column selectively using acid, base, thiocyanate, or a high salt such as magnesium chloride. The eluted antibody is then neutralized and dialyzed to remove the salts. Such antibody usually represents only ~1% of the total IgG in the serum. This procedure is different from the so-called affinity-purified antibody in which protein A or protein G is used; the latter purifies IgG only and has no meaning in relation to selective antigen reactivity.

Production of Polyclonal Antiserum The following procedure can be used to produce polyclonal antiserum in rabbits (Beltz and Burd, 1989). Preimmune blood is removed from the rabbit’s ear vein for later use in control experiments. The rabbit is immunized with of the antigen. If a carrier protein is used, the carrier and the antigen are injected together. One milliliter of the antigen (including the carrier) in buffer and 1 ml of Freund’s complete adjuvant are emulsified completely and injected subcutaneously into several locations on the rabbit’s back. Freund’s complete adjuvant contains ingredients that increase the rabbit’s immune response. The complete adjuvant includes saline, emulsifying agent, mineral oil, and killed mycobacteria. The mixture is injected once a week for 3 weeks, and then the animal is maintained for 3 weeks without additional injections. Approximately 25–40 ml of blood is removed from the animal’s ear vein to test for antibodies. One week after the first bleed the rabbit is boosted with half the antigen amount used earlier along with incomplete adjuvant; incomplete adjuvant does not contain the mycobacteria. Serum is again removed 2 weeks after this injection and tested for antibody response. The enzyme-linked immunosorbent assay is used to determine if the titer (or antibody concentration) of the serum is sufficiently high to establish antibody binding to the antigen. This 3-week cycle is repeated as long as necessary to obtain the antibodies desired. The peak response is generally achieved at the sixth to eighth injection (~5–7 months) after the initial immunization. If the rabbit is to be sacrificed, the final bleeding can yield 50–70 ml of serum. When the animal has made antibody in sufficient quantity, its serum can be used directly as the immunohistochemical reagent. It can be purified using an affinity column to remove nonimmunoglobulin proteins, extraneous antibodies, or to select antibodies that recognize a specific antigen. The use of affinity purified antibodies reduces nonspecific, background staining.

Affinity Chromatography Antibody affinity chromatography is employed to isolate antigen-specific antibodies. The most common affinity matrix for coupling of molecules is cyanogen bromide–activated

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Sepharose. The following procedure can be used to purify antibodies raised against a particular protein (Javois, 1999). 1. Sprinkle 1 g of cyanogen bromide–activated Sepharose 4B (Pharmacia-LKB, Piscataway, NJ) over 20 ml of 10 mM HC1. The gel swells immediately. One gram of dry gel yields ~3.5 ml of hydrated matrix. 2. Wash the gel on a 50-ml coarse, sintered glass funnel four times with 50 ml of 10 mM HCl by repeatedly suspending the matrix in the HCl solution, and then drain using vacuum suction. These washing steps remove the additives present in the dry matrix. 3. Suspend 5 mg of rat immunoglobulin in 5 ml of coupling buffer A (100 mM 500 mM NaCl, and 200 mM glycine, pH 8.0). 4. Add this supension to the gel and mix by inversion overnight at 4°C in a capped 15-ml polycarbonate tube. Avoid mechanically stirring the gel to avoid damaging the gel matrix. 5. Pour the matrix into a sintered glass funnel and drain the gel; save the eluate to estimate the amount of antibody couple. Estimate the amount of protein bound to the column by subtracting the quantity of IgG that is eluted. The eluate should not contain more than 20% of the applied protein concentration. 6. Wash the matrix with 100 ml of coupling buffer A to remove any unbound ligand. 7. Suspend the matrix overnight at 4°C in 45 ml of 200 mM glycine (pH 8.0) and mix by inversion in a 50-ml capped tube to block any unreacted groups. 8. Drain and wash the gel with three cycles of alternating pH. First, suspend the drained gel in 50 ml of 100 mM sodium acetate (pH 4.0) and 500 mM of NaCl. Drain with vacuum suction and wash with 50 ml of coupling buffer A. Drain and repeat the alternating pH washes twice. 9. Suspend the gel in 20 ml of BBS buffer (dissolve 247.3 g of boric acid, 187 g of NaCl, and 75 ml of 10 M NaOH in 4 liters of distilled water, pH 8.0). The matrix is then ready for use in column chromatography. 10. Pack the matrix in a Poly Prep column (Bio-Rad), which can be stored at 4°C; it should not be allowed to warm up or dry out. 11. Attach the column outlet to a peristaltic pump, and wash the column with 5 ml of BBS at 0.5 ml/min. 12. Drain most of the BBS, leaving ~0.5 ml on top of the gel bed. 13. Apply 15 ml of rabbit antirat IgG to the column, and circulate the solution through the matrix at 0.2 ml/min for 3 hr at 4°C. 14. Drain the column as in step 12 and save the eluate, which may still contain some of the desired antibodies. The titer of the eluate can be tested for reapplication to the gel at the end of the first purification, although this may not be necessary. 15. Wash the matrix with ~10 column volumes of BBS until the absorbance of the eluate is ) or using high concentrations of a cryoprotectant. However, the former is difficult to attain, and the latter tends to cause chemical toxicity and high osmotic stress. Because biological specimens possess low thermal conductivity and high thermal capacity, ultrarapid cooling can be obtained only for very small specimens. The above-mentioned difficulties in obtaining ultrarapid freezing can be minimized in the presence of microwave heating. This treatment suppresses ice crystal formation near the specimen surface, thereby extending the depth of good freezing from the specimen surface. Another advantage is better reproducibility of results because the state of water near the specimen surface is under control with microwave heating. Two mechanisms responsible for decreased rate of ice crystal growth are suggested. It is possible that the electric field component of electromagnetic radiation interacts with dipolar water molecules, disrupting the ice nucleation phenomenon (Hanyu et al., 1992). In other words, microwave heating reduces the size and number of ice crystal nucleation centers near the specimen surface. An alternative explanation is based on microwave radiation interfering with the kinetic processes of ice crystal growth (Jackson et al., 1997). For an ice crystal to form and grow, each water molecule must have an appropriate spatial orientation, position, and energy. Rapid ice crystal growth requires the molecular clusters to share edges and faces with the ice lattice without the induction of mutual strains. The torques produced by a microwave field can increase the number of available isomeric configurations, reducing the likelihood of a cluster of molecules having a configuration suited to integrating into a crystal lattice. Further development in the application of microwave heating to vitrification of biological specimens is awaited. Two apparatuses have been constructed for achieving ultrarapid freezing in the presence of microwave heating (Hanyu et al., 1992; Jackson et al., 1997). Microwave treatment can be employed with or without a cryoprotectant. According to one method, microwave heating is used at 2.45 GHz for a short duration (50 msec) immediately before and during tissue contact with the surface of a copper block cooled with liquid nitrogen (Hanyu et al., 1992). The ultrastructure is well preserved to a depth of from the contact surface, which is comparable to the depth obtained by the metal contact method using liquid helium in the absence of microwave heating.

PARAFFIN EMBEDDING For routine immunohistochemestry, surgical and other tissues are embedded in paraffin which is a mixture of hydrocarbons. Automated paraffin tissue processors are commercially available that customize the schedule to meet specific needs. However, tissues of

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various types and sizes as well as the particular study objective require optimal conditions of dehydration, infiltration, and embedding in paraffin. Chemical and physical changes occur in specimens during these treatments, affecting the sectioning and immunostaining qualities. Longer than optimal durations of these steps is a common habit, resulting in hard and brittle tissues that are difficult to section. It should be noted that additional fixation occurs during dehydration, accompanied by antigen masking and lipid dissolution. After fixation, a series of ethyl alcohol (a water-miscible solvent) of ascending concentrations is used to remove water from the tissue. Free water from the tissue is easily removed by diffusion. Water attached to the tissue by hydrogen bonds is also replaced by ethyl alcohol of higher concentrations. The efficacy of a solvent depends on its hydrogen bonding strength and molecular weight (Wynnchuk, 1993). Higher temperatures, vacuum, and microwave heating expedite the speed of dehydration, allowing shorter durations of dehydration. Xylene (an aromatic hydrocarbon) is used to replace ethyl alcohol from the tissue before infiltration with paraffin. Xylene is miscible with ethyl alcohol and paraffin. Xylene is called a clearing agent because it has a refractive index similar to that of proteins and thus renders tissue more or less transparent. It is generally satisfactory when the tissue blocks are not thicker than 3–4 mm. Xylene must be completely removed with paraffin, otherwise tissue will not section. Excessive exposure to xylene causes further denaturation of tissue proteins, causing difficulties in sectioning. Some lipid extraction also occurs in the presence of xylene. The treatments mentioned above to expedite the diffusion of ethyl alcohol also speed up the penetration of xylene. Xylene is also used between ethyl alcohol and mounting sections with resinous mounting medium after staining. The volatility and inflammability of xylene render it potentially dangerous. It must be used in a fumehood. While tissue is in xylene, gradual infiltration with paraffin is carried out. For tissues of a small size, 2 to 3 hr of paraffin infiltration is adequate. For large tissues (5–10 mm), overnight infiltration is required. The temperature during infiltration must not be higher than 4° above the melting point of paraffin (54–58°C). Vacuum embedding can be carried out to remove air bubbles from the tissue and rapidly replace the clearing agent with paraffin. This approach is especially desirable for aircontaining tissues such as lung or hard tissues such as fibrous or scar tissues. The vacuum should not exceed 400–500 mm of mercury to avoid damage to the tissue. Paraffin blocks are trimmed with a scalpel, a razor blade, or a hot spatula and mounted on wooden or fiber blocks. While being sectioned, longitudinal block edges must be parallel to the knife edge to obtain a ribbon. Paraffin sections of any thickness show compression, which is usually relieved when they are floated on a glass slide and dried. It should be noted that the melting point and crystalline structure of paraffin influence the section quality. Paraffin of a lower melting point is less brittle when solidified; however, it tends to show more compression during sectioning. On the other hand, paraffin of a higher melting point provides a better support for hard specimens. Paraffin of a smaller crystalline structure adheres closely to the cell components in the embedded tissue, providing good support for sectioning. Paraffin sections of a larger crystalline structure show more pronounced curvature, which is difficult to flatten after sectioning. To obtain crystals of a small size, paraffin should be cooled rapidly. Deeper layers of the tissue block contain larger and looser crystals, resulting in poor quality of sections in these layers.

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Paraffin Embedding in Microwave Oven Recently, a new type of microwave oven (HFX-800, Meditest, Illatos ut 9, 1097 Budapest, Hungary), combining microwave heating and vacuum, was introduced (Kovacs et al., 1996). The oven is reported to allow fixation, dehydration, paraffin embedding, and section staining. The histoprocessing is completed in depending on the thickness of the tissue block. Decalcification of the bone specimen can also be carried out in this oven. The power level can be switched from 10 to 650 W by the chosen cycle time. The temperature can be preset and subsequently controlled automatically between 0 and 120°C. Vacuum is generated by a built-in vacuum pump (operating at 12 V), producing 0.0–0.3 bar vacuum. The vacuum level is indicated by an automatic meter. Since the oven provides a sealed and ventilated system, the evaporation of formalin, ethanol, isopropanol, and other reagents does not affect the operator. Vapors and fumes are extracted by continuous ventilation. The oven weighs about 21 kg. Its use is awaited.

Paraffin Embedding in Vacuum-Microwave Oven Vacuum combined with microwaving has been tried for embedding the tissue in paraffin, using Milestone’s MicroMED LAVIS-1000 machine (Marani et al., 1996; Bosch et al., 1996). The advantage of this system is that microwaves travel with ease through a vacuum, whereas conventional heating under vacuum is difficult. This machine provides pressure reaching 100 hPa, and the microwave oven attains a maximum power of 1,000 W; its cycle time can be adjusted between 0.1 and 0.5 sec. The machine is equipped with an infrared temperature probe which allows temperature control from outside the unit. To obtain satisfactory results, coordination of temperature with vacuum is necessary. Paraffin embedding is carried out in a stepwise descending series: 700 hPa, 500 hPa, 300 hPa, and 100 hPa. A too-rapid lowering of the pressure is damaging to tissue morphology. The temperature during dehydration with isopropanol should not exceed 60°C. Further improvements of this system are awaited.

Microtomy of Paraffin-Embedded Tissues Commercial paraffin is a mixture of a straight chain of hydrocarbons that contain additives. Both the melting and the plastic points of paraffin are related to the sectioning properties. The plastic point occurs ~10°C below melting point. The role of the melting point becomes apparent when one considers that the higher-melting-point hydrocarbons crystallize first as flat plates that accumulate on one another as successively lower-meltingpoint hydrocarbons crystallize (Allison, 1998). These dynamic processes force the plates to curl and roll, giving rise to needle-shaped crystals. Needle-shaped crystals are considered ideal for microtomy. Thus the proportion of plates and microcrystals depends on the proportion of high-melting-point and low-melting-point hydrocarbons in the paraffin. The shape and the size of crystals are influenced by the nature of cell and tissue structures as the molten paraffin infiltrates and solidifies in the tissue spaces.

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Two types of forces are exerted during paraffin sectioning: flow shearing and pointto-point shearing (Allison, 1998). Flow shearing proceeds ahead of the cutting edge, resulting in smooth sections. In contrast, point-to-point shearing travels through the path of least resistance ahead of the cutting edge, producing a section of uneven thickness. Paraffin contains additives that minimize the point-to-point shearing and reduce the plastic flow. Additives are synthetic polymers that improve the consistency of paraffin by filling the spaces among paraffin crystals in the tissue. Cut a paraffin block containing one tissue specimen with a razor blade, and mount it to a support stub. Trim the block manually with a razor blade or on an automatic trimming microtome to a rectangular or trapezoidal cutting face. The size of the block face is determined by the objective of the study and the size of the tissue specimen. The upper and lower edges of the block facing the knife cutting edge should be parallel to each other to obtain a ribbon, if required. Mount and orient the block on the microtome, so that its longer edge is parallel to the cutting edge. A steel or glass knife can be used. Cut sections ( thick) on a rotary microtome, which usually has an automatic advance mechanism that can be set to advance the specimen block the desired distance toward the knife with each stroke. Manual or motorized rotary microtomes are commercially available (Triangle Biomedical Sciences, Durham, NC; Sakura Finetek, Torrance, CA). A microtome with automated specimen approach, trimming, and sectioning is also available (Leica Microsystems, Deerfield, IL). Float the sections on water or 4% formalin on a glass slide and heat for 10–15 min at ~40°C on a warming plate to remove the compression. Remove the liquid with a fine pipette and dry overnight in an oven at ~40°C to ensure section adherence to the slide. Drying can also be accomplished in 15 min at ~40°C in a microwave oven. Note: Sectioning will be adversely affected if tissue infiltration with paraffin is incomplete. A too-shallow or too-steep bevel angle of the steel knife relative to the tissue block face will result in section compression and chatter, respectively. The optimal cutting angle is 4°. Section adhesion to the glass slide can be ensured by coating the slide with polylysine ( to 1 mg/ml) in 10 mM Tris (pH 8.0). Alternatively, coated slides are commercially available (Probe-On-Plus slides from Fisher Scientific). Make sure that the tissue specimen is firmly mounted to the stub and the latter to the microtome. Also, the paraffin block should be fairly cold at the time of sectioning. Low humidity in the vicinity of the microtome tends to result in static electricity, which makes it difficult to separate the section from the block face after cutting. For additional details, see Ruzin (1999).

Silanting of Glass Slides

Detachment of tissue sections from glass slides during processing for immunohistology is not uncommon. Such detachment can be avoided by preparing silanted slides as follows (Miksys, 1999). 1. 2. 3. 4.

Wash slides in common household detergent. Rinse in running tap water for 10–15 min. Rinse in distilled water. Rinse in 100% acetone for 5 min.

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5. Coat with 2% Aplex (3-aminopropyltriethoxysilane) (Sigma) in 100% acetone for 5 min. 6. Rinse in lightly warm running tap water for 2 min. 7. Rinse in distilled water. 8. Dry at ~40°C in a dust-free area. 9. Store at room temperature up to 1 month or at –20°C for several months. Freshly coated slides are preferred.

Vacuum-Assisted Microwave Heating A vacuum–microwave combination has been used for processing tissues for light microscopy (Kok and Boon, 1996), transmission electron microscopy of animal tissues (Giberson, 2001) and botanical specimens (Russin and Trivett, 2001), and scanning electron microscopy of human lymphocytes (Demaree, 2001). The vacuum-microwave heating method is especially useful for processing botanical tissues because these specimens possess physical characteristics that hamper easy penetration of reagents; these characteristics include cell wall, vacuoles, plastids, and intercellular spaces. Secondary cell walls may contain cutin, suberin, and lignin, which are hydrophobic. These waxy substances limit the evaporation of water from the tissue and resist the penetration of reagents. The presence of air in the intercellular spaces creates a barrier to fixative penetration. These impediments to reagent penetration and action of fixatives can be significantly reduced by using vacuum–microwave heating. For details of this methodology, see Russin and Trivett (2001).

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Chapter 4

Factors Affecting Antigen Retrieval

Many factors influence antigen retrieval, including fixation, heating, retrieval fluid, and antibodies.

FIXATION Fixation is the most important factor affecting antigen retrieval. Type of fixative and duration and temperature of fixation are all important. Many varieties of epitopes have been retrieved with various degrees of success in tissues fixed with formalin, methanol, methacarn, or Bouin’s fixative; buffered 10% formalin containing 3.7–4.0% formaldehyde is the most commonly used. Although fixation with paraformaldehyde or glutaraldehyde better preserves cell morphology because of stronger, and more rapid and more extensive protein crosslinking, antigen unmasking becomes difficult. The use of formalin has become a matter of habit and convenience, especially in pathology laboratories; it is also inexpensive. To improve the preservation of cell morphology, it is recommended that a mixture of formalin (or paraformaldehyde) and glutaraldehyde (0.05–0.5%) be tried. Such mixtures are routinely employed for immunocytochemical studies with the electron microscope. It is known that some types of antigens are resistant to fixation with low concentrations of glutaraldehyde. Preembedding immunocytochemistry by the avidin-biotin method, which avoids fixative effects, has been successfully applied for identifying peptide or protein antigens in the brain tissue fixed with glutaraldehyde (Mrini et al., 1995). The effect of fixation on antigenicity is complex. With the exception of a minority of antigen types (e.g., PCNA nuclear protein) that are formalin resistant to various degrees, most antigens are sensitive to the concentration of the fixative and the duration of fixation. Although 10% formalin is the usual fixative, it is inadequate for preserving some types of antigens that are fixative resistant. It has been demonstrated histochemically, for example, that PCNA nuclear protein antigenicity is preserved much better with 20% formalin-PBS than with 10% of the same fixative (Muñoz de Toro de Luque and Luque, 1995). The reason is that the protein antigen is partially extracted with the lower fixative 71

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concentration. In this respect, it should be noted that some types of antigenicity, such as PCNA, require quantitative measurements for assessment of clinical significance, as its low levels may be present in quiescent cells. Is antigenicity affected by factors other than fixation? Yes. Although fixation is the most important factor in tissue processing for immunohistochemistry, other factors tend to affect immunorecognition of antigens. These factors include the interval between removal of the tissue from the human or animal and fixation; the method of excising the tissue from the body (mechanical damage); the technique of cutting sections of the tissue embedded in paraffin or resin; the procedures for removing section compression, folds, or bubbles and attaching it to the glass slide; the interval between cutting sections and immunostaining (storage or without storage of slides prior to staining); and other immunostaining details. Folds and bubbles hinder section adhesion to the slide, and they may also show 3,3-diaminobenzidinetetrachloride (DAB) precipitation. Bubbles under sections may appear as brown spots on immunostained sections (Grizzle et al., 2001). Paraffin sections ( thick) adhere tenaciously to glass slides by heating overnight at 65°C. The use of a PAP pen or other means to demarcate the tissue to aid in staining is also a variable. Awareness of the above-mentioned variables should prevent erroneously attributing them to problems with fixation. Tissue specimens ideally should be placed in the fixative immediately after their removal from the body. This problem arises in studies of human tissues, for their immediate fixation is usually not feasible. If immediate fixation is not possible, the tissue must be kept cool and moist by covering it with a piece of cloth soaked in sterile, cold saline for not more than 20–30 min. During this time the specimen should not contact any dry and absorbent object such as paper, a paper towel, or gauze. To keep the paper trail of the specimen (source, time, place of collection, etc.) is no less important. Note that even human tissues fixed immediately after their removal from the body may undergo cellular changes because usually the vascular supply is terminated before the tissue is surgically removed. During this duration (~1 hr) the tissue remains at body temperature, at which the activity of digestive enzymes continues, damaging the cellular structures (Grizzle et al., 2001). Chemical fixation is not the only factor that causes loss or irreversible masking of antigens. Treatments such as dehydration and embedding following fixation also play a role in the loss of immunorecognition of antigens. Absolute ethanol and xylene must not contain traces of water, and the water bath should be very free from contaminants such as bacteria, fungi, dust, and dirt. Once the sections are contaminated, they cannot be decontaminated. According to Watanabe et al. (1996), the antigen preservation test (Riederer, 1989) showed that immunostaining intensity, for example of decreased during fixation with paraformaldehyde but did not decrease during washing and immunostaining. The proportionate decrease in intensity due to fixation was almost constant even when the amount of the antigen differed in the sections. They concluded that the decrease in immunostaining intensity was related to a proportional decrease in antibody binding due to masking of antigens during fixation.

DENATURATION On the basis of antigen retrieval obtained with protein denaturing agents, it has been proposed that in certain cases antibodies recognize denatured but not native antigens.

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However, this proposition seems untenable, given the requirement of a specific amino acid sequence for an epitope, as well as a specific conformation of antibody molecule, in order for antigen-antibody binding to occur. How could an antibody react with a completely denatured antigen, when the former is usually generated using the native form of the latter? For example, antibodies generated against selected regions (N-terminal fragment or C-terminal region) of corresponding antigens recognize predominantly similar undenatured regions, unless these regions of the antigen are masked by other regions of the antigen and/or by some surrounding components. The proposition that antibodies recognize certain antigens only after the latter have been denatured is true only when the epitope is unmasked and remains undenatured following antigen denaturation. In other words, a denatured epitope cannot be recognized by the antibody if the amino acid composition of the epitope peptide and/or its linear amino acid sequence is altered or damaged. The reaction between the antigen and the antibody is dependent on the conformation of the former. However, the presence of an intact threedimensional folded antigen structure may not be necessary in certain cases for antibody binding. Denaturation or unfolding of certain antigen molecules may be necessary to unmask the epitope that is buried in the interior of the folded antigen structure (personal communication, Dennis Brown). It is likely that most antigen molecules form multiprotein complexes, resulting in masking of epitopes by surrounding proteins. The epitope masking becomes more serious when these complexes are crosslinked with formaldehyde. Such masked epitopes can be recognized by the antibody only when exposed by breaking crosslinks and denaturing surrounding cell components. If this is so, denaturing treatments cause breakdown of reversible crosslinks introduced by formaldehyde and denaturation of surrounding cell components, enabling the antibodies to recognize the native, uncrosslinked or partially crosslinked undenatured structure of the reactive epitope molecule. It means that denaturing treatments do not denature antigen per se but denature multiprotein complexes of which the antigen is a part. It may be that imprecise nomenclature has given rise to confusion in this field. It should also be noted that denaturing agents make cells permeable, facilitating antibody penetration. Moreover, these agents are used usually in combination with microwave heating. Therefore, the role of these agents in epitope retrieval needs to be explained in the context of their role in cell permeabilization as well as of the influence of elevated temperatures.

HEATING Different heating methods, including microwaving, autoclaving, pressure cooking, microwaving combined with pressure cooking, steam heating, and water bath heating, have been employed for antigen retrieval with various degrees of success. However, microwave heating, introduced by Shi et al. (1991), is most commonly used for retrieving a wide range of masked antigens in formalin-fixed and paraffin-embedded tissues. Heating at a high temperature (100°C) for a short duration (10 min) gives better results than those achieved with a comparatively low temperature for a longer time. However, there are some exceptions. According to Evers and Uylings (1994a), the immunostaining of SMI32 obtained at 90°C was superior to that achieved at full power heating.

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pH Another important factor in achieving optimal antigen retrieval is the pH of the retrieval solution. It is thought that the pH is more important than the constituents of the retrieval fluid (Shi et al., 1995a). There is, however, no universally optimal pH for a retrieval fluid. The retrieval of most types of antigens requires a specific pH, although retrieval of a few antigens can be achieved over a wide range of pH levels; for example, AE1 and NSE (cytoplasmic antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens) can be retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, following retrieval at pH 3–6, some antigens (e.g., estrogen receptor) display a marked decrease in the immunostaining, while still other antigens such as cytoplasmic HMB 45 show weak or negative staining after retrieval at pH 1–2 but excellent results in the high pH range (Shi et al., 1995a). Some antigens can be retrieved only at a low pH. These types are exemplified by thrombospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and 2.5, respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). However, pH levels lower than 3.0 can severely damage tissue morphology. Low pH levels can also alter the localization of some cytoplasmic antigens, resulting in false-positive staining of the nucleus. For example, an antibody (UCHL1) to T cell antigen is effective at pH 6.0 but results in the staining of every nucleus at pH 2.0 (personal communication, H.Y. Lan). In summary, the use of sodium citrate buffer at pH 6.0 increases the intensity and extent of immunostaining of a wide variety of tissue antigens, whereas Tris-HCl buffer may yield better results for some antigens at pH 10.0. On the other hand, low-pH antigen retrieval fluids are necessary for some antigens such as thrombospondin. For previously unexamined antigens, a test battery based on three pH values (low, middle, and high) should be carried out to establish an optimal protocol (Shi et al., 1996a).

MOLARITY The concentration of antigen retrieval fluid is often less important than temperature, duration of heating, and pH in achieving optimal antigen retrieval. For example, sodium citrate buffer is effective at molarities ranging from 0.01 to 0.5. However, in the case of another antigen retrieval solution, ammonium chloride, 0.5%, 1%, 2%, and 4% solutions were tested, and 4% concentration yielded the best immunostaining of vimentin in archival paraffin sections (Suurmeijer and Boon, 1993a). Ammonium chloride solutions are weakly acidic (pH 3–4). According to Bruno et al. (1992) and Muñoz de Toro de Luque and Luque (1995) minor changes in ionic strength affect the PCNA nuclear protein antigenicity involved in DNA synthesis. If enzyme digestion methods are used, the optimal concentration of the enzyme (e.g., protease) must be applied. Unlike sodium citrate buffer, enzyme solutions cannot be used at a range of concentrations. Note that the concentration of the diluent used for primary monoclonal antibodies does affect the specificity and intensity of immunostaining (see page 82). It should be noted that the concentration of the antibody also affects the specificity and intensity of immunostaining (see page 80).

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ANTIGEN RETRIEVAL FLUIDS Several antigen retrieval fluids are in use, all having been reported to efficiently mediate antigen retrieval. A fluid applicable to all antigens is not available. The main reason is the enormous variety of chemical structure of not only antigens but also epitopes of any one antigen. In fact, the chemical nature of the epitope plays a key role in the effectiveness of an antigen retrieval fluid. In other words, the tissue, cell, and antigen types determine the retrieval fluid. This is substantiated by the fact that generally each type of epitope determines the fluid type conducive to its maximal retrieval. A few examples follow. Two antigen retrieval fluids, sodium citrate buffer (0.1 M, pH 6.0) and glycine-HCl buffer (0.05 M, pH 3.5) containing 0.01% EDTA, were compared for their effectiveness in unmasking a wide variety of antigens (Imam et al., 1995). Glycine-HCl buffer-EDTA yielded stronger immunostaining of p53, androgen, estrogen, progesterone, and Ki-67, whereas sodium citrate buffer produced superior immunostaining of vimentin and leukocyte antigens. PCNA was unmasked equally well with either of the two antigen retrieval buffers, while the two buffers were ineffective in retrieving antigens such as prostatic acid phosphatase and pan-keratin. According to another study, compared with sodium citrate buffer, Tris-HCl buffer (pH 9.5) containing 5% urea yielded more intense staining of Ki-67 in mouse lung tumors (Ito et al., 1998). However, low background staining is likely when using the latter antigen retrieval buffer. Certain other types of antigens require a combination of antigen retrieval fluids or systems for their optimal retrieval. Methods using such combinations are given in this volume. Comparative effects of antigen retrieval systems on antigens are summarized in Chapter 6. In addition to the chemical structure of antigens, a number of other factors, including pH, heating temperature, molarity, and the chemical composition of the retrieval fluid, are considered for selecting the optimal retrieval fluid. Optimal immunostaining of a given antigen requires an antigen retrieval fluid of a specific pH. Note that optimal antigen retrieval requires an optimal fixation procedure. Although the exact mode of action of antigen retrieval fluids is not known, their salts may modify the hydrophobicity of polypeptide chains, affecting the conformation of protein molecules. The major effect of the salts is to mediate high temperature effects. However, this mechanism does not explain the mode of action of nonbuffer fluids (e.g., water) used for antigen retrieval. Excellent p53 immunostaining in breast tumors has been achieved by heating the sections in water for 15 min at 50°C (Katoh and Breier, 1994). Heating is carried out in a microwave oven or in a water bath. Antigen retrieval can occur under both acidic and alkaline conditions, depending on the type of antigen involved. The mechanisms involved in the antigen retrieval at different pH values are not known. Three commonly used antigen retrieval fluids are 0.01 M sodium citrate buffer (pH 6.0), 0.01 M Tris-HCl buffer (pH 1.0) or 0.1 M Tris-HCl (pH 10.0), and 0.05 M glycine-HCl buffer (pH 3.6). The latter can be used with or without 0.01% EDTA depending upon the antigen type. Taylor et al. (1996b) recommend 0.1 M Tris-HCl buffer (pH 9.5) containing 5% urea. These fluids provide strongly alkaline or acid environments and are effective for antigen retrieval in tissues which have been either mildly fixed or overfixed with formalin. These recommendations are based on the successful immunostaining of a wide variety of antigen-antibody complexes. For most clinical applications, 0.01 M sodium citrate buffer (pH 6.0) is recommended.

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Only if sodium citrate buffer or Tris-HCl buffer fail to yield satisfactory retrieval should fluids containing substances such as EDTA, EGTA, enzymes, metal salts, periodic acid, or urea be tried. Consider the immunostaining of parvalbumin, calbindin, and MAP, which has been found to be best accomplished using 4% aluminum chloride (Evers and Uylings, 1994b). However, in this study the antigen retrieval effect of the metal solution was compared only with that of distilled water and zinc sulfate; sodium citrate buffer was not tested. Since free-floating vibratome sections of the human brain tissue underwent severe wrinkling during microwave heating, thick tissue slices (0.5 cm) were placed in 4% aluminum chloride solution and heated in a microwave oven for 10 min, followed by standard immunocytochemical staining of semithin sections Another advantage of pretreatment is that the brain tissue hardens, facilitating easier sectioning. Fluids other than standard sodium citrate and Tris-HCl buffers are also preferred in some other cases. An example is the retrieval of Bcl-2 antigen (oncoprotein), which is best achieved by hydrated autoclaving of sections placed in deionized water (Umemura et al., 1995). Immunostaining of neurofilament proteins, proliferating cell nucleus antigen (PCNA), retinal S-antigen, and glial fibrillary acidic protein (GFAP) has been obtained by using distilled water as the antigen retrieval fluid in a microwave oven (Yachnis and Trojanowski, 1994). However, heating in water in a microwave oven is not generally recommended. Neurofilament proteins in archival tissues have been immunostained after employing a saturated solution of lead thiocyanate (Yachnis and Trojanowski, 1994). Zinc sulfate has been used for retrieving vimentin and prostate-specific antigen (Wieczorek et al., 1997). Cesium chloride (5.7 M) has also been employed for antigen retrieval. However, such metal salt solutions are not recommended because they are toxic. Target unmasking fluid (TUF) was developed by van den Berg et al. (1993) for routine immunohistochemistry and is commercially available (Signet Lab, Delham, MA, or Kreatech Biotechnology, Amsterdam). Periodic acid (0.5%) has also been used as an antigen retrieval fluid (Xue et al., 1998). Another type of antigen retrieval fluid is EDTA of pH 8.0 (Morgan et al., 1994; Pileri et al., 1997), which has been stated to be effective irrespective of the location of the target molecule (intranuclear, intracytoplasmic, or membrane-bound). Comparative studies by Ehara et al. (1996) also indicate that EDTA (0.15 M, pH 6.0) yields stronger immunostaining of steroid hormone receptors than that obtained with sodium citrate buffer (0.01 M, pH 6.0). However, the preservation of cell morphology is superior when citrate buffer is used. Urea (3 M), formic acid, and guanidine solutions have also been employed for antigen retrieval. When urea is used in an autoclave or a pressure cooker, it has the disadvantage of yielding false-negative results or background staining (Shi et al., 1996b). In another study, a saturated solution of dimedone was applied for antigen retrieval (Shi et al., 1996b). Recently, it was reported that the addition of calcium chloride to the antigen retrieval fluid of a low pH improved the preservation of tissue morphology (Morgan et al., 1997a,b). Boric acid (0.2 M, pH 7.0) in conjunction with low-temperature, heat-mediated antigen retrieval technique has been successfully used as the antigen retrieval fluid for estrogen receptors on freshly cut sections of breast tissue (Peston and Shousha, 1998). Boric acid is also very effective in antigen retrieval on the archival hematoxylin-eosin-stained lymphoid sections on coated or uncoated slides, using conventional heat-mediated antigen retrieval method (Biddolph and Jones, 1999). Lymphoid sections tend to dislodge from the coated or uncoated slides in the presence of sodium citrate buffer during antigen retrieval.

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The above discussion indicates that although 0.01 M sodium citrate buffer (pH 6.0) is commonly used, it is not a universally ideal antigen retrieval fluid for all types of tissues and antigens. If published information is not available with regard to the best antigen retrieval fluid for the antigen under study, the ideal retrieval fluid for each type of epitope must be determined by trial and error. The following four antigen retrieval fluids are commercially available (BioGenex, San Ramon, CA). The approximate pH indicated below is valid at the time of manufacture; the pH of the fluid may change during storage. 1. 2. 3. 4.

Antigen Retrieval Citra Microwave Solution used at pH 6.0. Antigen Retrieval Citra Plus Microwave Solution used at pH 6.1. Antigen Retrieval Glyca Microwave Solution used at pH 3.5. Antigen Retrieval AR-10 Microwave Solution used at pH 10.5.

Other commercial sources for antigen retrieval fluids are: 1. Dako TRS, Dako Corporation, 6392 Via Real, Carpinteria, CA 93013 HIER buffer, Ventana Medical Systems, Tucson, AZ 2. Target Unmasking Fluid (TUF*), Monosan (Sanbio), Fronstraat 2A, Postbus 540, AM Uden, NL-5402, The Netherlands; Serotec Ltd., 22 Bankside, Station Approach, Kindlington, Oxford, Oxon OX5 1JE, U.K.

Glycerin as Antigen Retrieval Fluid When other antigen retrieval methods fail, antigen retrieval can be accomplished in 90% glycerin solution using a hot plate with a magnetic stir rod. This approach is thought to improve preservation of tissue morphology as well as efficient retrieval of some antigens. Glycerin has the advantage of having a very high boiling point (290°C) and being nontoxic, stable, and reusable. The stir bar maintains a constant and uniform temperature throughout the antigen retrieval fluid, prevents hot or cold spots, and thus facilitates reliable and consistent results. This method can also be used in a conventional hot air oven. Many slides in metal slide racks can be processed simultaneously in this oven. Glycerin solution can also be used for antigen retrieval in Coplin jars in a microwave oven; an empty space must be kept between the slides. The glycerin method has been used for retrieving a number of antigen types, including estrogen receptor (Beebe, 1999). It is especially useful for very small, fragile biopsies such as prostate needle biopsies and bowel biopsies. Pure glycerin fails to bring about antigen retrieval, which means that water and heat are required to cleave the formaldehyde molecule from the proteins, break down the methylene bridges, or rehydrate the proteins. The exact role of glycerin in the antigen retrieval mechanism is not known. Further testing of the usefulness of this procedure is awaited.

*Contents: chromium potassium sulfate dodecahydrate, sodium dodecyl sulfate, dextran sulfate, formamide, phosphate salt, magnesium sulfate, pepsin, polyethylene glycol, and Triton. It has a low toxicity and is irritating to the eyes and skin. It is a colorless, nonviscous liquid.

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Procedure

The antigen retrieval fluid consists of a mixture of 100 ml of 90% glycerin and 10 ml of Dako’s citrate buffer. The slide rack is placed in a bowl of an appropriate size and shape, so that the stir bar rotates freely under the slide rack. A sufficient volume of antigen retrieval fluid is transferred to the bowl ~ 1 cm above the level of the slides. The hot plate is turned on and adjusted until the temperature of the fluid reaches 100–120°C; the duration of heating varies between 5 and 20 min. For estrogen and progesterone retrieval the duration of heating (120°C) is ~7 min with a 15-min cool-down period before the slides are transferred to distilled water for further processing. Alternatively, such high temperatures in the bowl can be achieved by placing it in a microwave oven, then removing and placing it on the hot plate. This is followed by adding the stir bar and the slide rack to the bowl. It takes 1 min for 100 ml of the glycerin solution to attain a temperature of 125°C in a 600 W microwave oven on high. If more than 100 ml of solution is to be heated to the same temperature, for every additional 100 ml an additional 1 min is required.

pH of Antigen Retrieval Fluids In addition to heating, retrieval fluid pH plays a key role in achieving optimal antigen retrieval. It is thought that the pH is more important than the composition of the retrieval fluid. This is supported by the demonstration that optimal staining of antigen SMI-32 was achieved at pH 2.5 and 2-hr microwave heating at 90°C, whereas staining of antigen MAP2 was best obtained at pH 4.5 and 10-min full-power heating; in both cases 0.05 M citrate buffer was used (Evers and Uylings, 1994a). Therefore, in optimizing the antigen retrieval protocol, pH is a priority. Note that there is no universally optimal pH for a retrieval fluid. The retrieval of each type of antigen requires a specific fluid pH, although exceptions occur with antigens that can be retrieved at a wide range of pH levels. For example, AEI and NSE (cytoplasmic antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens), can be retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, some antigens (e.g., estrogen receptor) display a marked decrease in immunostaining at pH 3–6, while still other antigens, such as cytoplasmic HMB45, show weak or negative staining at pH 1-2 but excellent results in the high pH range (Shi et al., 1995a). Some antigens are retrieved only at a low pH. These types are exemplified by thrombospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and 2.5, respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). Note, however, that pH levels lower than 3.0 can severely damage tissue morphology, especially with intense heating. Low pH levels can also alter the localization of some cytoplasmic antigens, resulting in false-positive staining of the nucleus. For example, an antibody (UCHL1) to T cell antigen is effective at pH 6.0 but results in the staining of every nucleus at pH 2.0 (personal communication, H. Y. Lan). Generally Tris-HCl buffer produces better results at higher pH levels (e.g., pH 10.0) than do some other buffers. On the other hand, sodium citrate buffer increases the intensity and extent of immunostaining of a wide variety of tissue antigens at pH 6.0. EDTA-NaOH (1 mM) at pH 8.0 also yields satisfying results. Although relatively high pH solutions, such

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as sodium citrate or Tris-HCl, are suitable for most antigens, low pH solutions are preferred for nuclear antigens (Taylor et al., 1996a). However, solutions of low pH generally tend to cause weak focal background staining and damage to some epitopes. A test battery based on three pH values (low, middle, and high) should be carried out to establish an optimal protocol, including pH, for immunostaining previously unexamined antigens (page 104).

Ionic Strength of Antigen Retrieval Fluids The ionic strength of the fluid in which tissues are suspended during fixation with formaldehyde, unlike retrieval fluid concentration, does influence antibody access to intracellular antigens such as proliferation cell nuclear antigen (PCNA), nuclear protein (Ki-67) detected by MIB-1 antibody, and nuclear antigen p120. Ionic bonds are known to be responsible for a major portion of protein-protein interactions, and their breakage causes dissociation of the interacting proteins, resulting in increased detectability of the antigen. Such breakage occurs with increased salt (NaCl) concentrations. It has been shown that the immunofluorescence of antigens such as PCNA is increased when the cells are fixed in the presence of increased salt concentrations (Bruno et al., 1992). The increase is greater for cells in the phase of the cell cycle than for cells in S or phase. High salt concentrations loosen the proteins, which are then stabilized with formaldehyde. In other words, increased ionic strength weakens intra- and intermolecular ionic interactions during the process of crosslinking with formaldehyde. Using the optimal ionic strength of the solution, which must be customized for a given antigen, will facilitate the accessibility of the antibody to the epitope.

ANTIBODY PENETRATION The fundamental question in the phenomenon of antigen retrieval is whether it is due to enhanced penetration of antibodies into the tissue or to reversal of protein conformational changes induced by fixation, or both. The evidence favors both explanations. All the treatments (microwave, autoclave, and conventional heating, enzyme digestion, ultrasound application, and detergent treatment) used for antigen retrieval break down protein crosslinkages, facilitating antibody access to the antigen. The achievement of increased immunostaining after using cell permeabilization methods testifies to the role of antibody penetration into the tissue. The above treatments also restore the original conformation of the protein molecule, resulting in enhanced interaction between the antigen and the antibody. Antibody molecules are relatively large. The speed and extent of antibody penetration into the tissue and the degree of fixation with formaldehyde are inversely related. In other words, the stronger the fixation (protein crosslinking), the slower the antibody penetration. The reason for this relationship is that extensive crosslinking results in the formation of a compact protein network which impedes antibody penetration. Such an impediment can be caused by the cell membranes as well as by the cytoplasmic matrix, both of which contain proteins. It is therefore apparent that strong, extensive protein crosslinking should be avoided before incubation with antibodies. This is the reason for preferring

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formaldehyde over glutaraldehyde, since the latter forms strong protein crosslinkages. It is also well established that tissue specimens that have been fixed for a long time (weeks or months) require more vigorous treatments of sections for antibodies to penetrate and have access to the antigens. It is interesting to note that an antibody against a specific epitope less sensitive to aldehyde fixation can be obtained by immunizing the mice with an antigen in which the aldehyde-sensitive epitope has been blocked or altered. To increase the penetration of antibodies into thin resin sections of fixed tissues, simultaneous heating of sections and antibodies has been attempted. This treatment is thought to increase the labeling of certain antigens, whereas that of some other antigens remains unaffected. It has been demonstrated that such a treatment enhanced labeling density by the antiamylase antibodies, whereas labeling with anti-DAMP antibodies remained unchanged (Chicoine and Webster, 1998). Further developments of this protocol are awaited.

ANTIBODY DILUTION Not only the type (e.g., the cell clone) and the source of availability of an antibody but also its dilution are important in fully utilizing the effectiveness of an antibody as a powerful tool to detect antigens. The optimal antibody concentration for antigen varies, depending on whether the tissue used is aldehyde-fixed or frozen; generally higher antibody concentrations are required for sections of aldehyde-fixed tissues (Fig. 4.1). Also, different forms of an antigen require different concentrations of the antibody for their maximal detection. This is exemplified by the PC-10 primary antibody, which identifies PCNA antigen at a dilution of 1:1000 in epithelial cells in normal colon tissue, whereas a dilution of 1:400 is required to localize these proliferating cells in adenomatous polyps (Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detected in various tissue types at the MIB-1 dilution of 1:50, using the microwave heating antigen retrieval method. However, in a few studies MIB-1 dilutions of 1:20 to 1:100 have been used. There are many pitfalls, including false-positive and false-negative staining, to using antibody concentrations of higher or lower than optimal concentrations. Labeling specificity partly depends on the antibody dilution, while background staining critically depends on its dilution. Also, the esthetic appeal of the images produced by immunohistochemistry diminishes with suboptimal dilutions of antibodies. Therefore, to avoid unwanted staining, pay careful attention to the optimal working dilution of an antibody, especially of a polyclonal antibody, and to washing procedures. Also note that antigen retrieval treatments allow the use of increased dilutions of the antibodies. For example, for sections of formalin-fixed and paraffin-embedded tissues, the optimal dilution of PC-10 antibody without heat pretreatment is 1:10 compared with 1:600 after microwave heating (Haerslev and Jacobsen, 1994). A fairly high concentration of the primary antibody is necessary to follow saturation kinetics. However, the majority of these antibodies exhibits a bell-shaped concentrationbinding curve, with the binding increasing up to a specific antibody concentration and then decreasing. Such a bell-shaped curve is due to unstable binding of the antibody to the antigen under very high antibody concentrations. Effects of a high antibody concentration can be examined with the method of Raivich et al. (1993). In practice, however, one rarely

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aims for or achieves saturation kinetics in routine immunohistochemistry. In most cases we aim for and achieve adequate and reproducible staining. High concentrations of primary antibodies can increase nonspecific binding and also compromise antigen-specific immunostaining. Furthermore, antibodies may aggregate at high concentrations, which limits their penetration. Electrical charges on aggregated antibodies may hinder their penetration among similarly charged cell molecules; therefore, antibodies should be used at concentrations at or slightly below antigen-specific concentrations. Note that similar antibodies obtained from different sources may not yield the same intensities of immunostaining. Some evidence indicates increased labeling efficiency of certain diluted antibodies (e.g., antiamylase antibodies and anti-MHC class II antibodies) when they are exposed to microwave heating prior to their application (Chicoine and Webster, 1998). However, such

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increased labeling may be accompanied by enhanced background staining. Furthermore, such a treatment reduces labeling density of some other antibodies such as anti-DNP antibodies. The duration of heating of antibodies is critical to obtain optimal labeling. The optimal heating duration varies with the antibody and the fixation parameters used to stabilize the cellular components. The mechanism(s) responsible for increased or decreased labeling efficiency of different antibodies as a result of their heating is not known.

Diluent Buffer for Primary Antibodies Both the pH and osmolarity of the diluent buffer (especially the type of solute) affect the affinity of monoclonal antibodies for the antigen. It is known that electrolytes exert a profound effect, not only on the structural relations of protein molecules, but also on the reactivity of proteins (Hayat, 2000a). The reactivity of both monoclonal and polyclonal antibodies with antigens is affected by the type of antibody diluent used. This is true whether or not a heat-induced antigen retrieval is used. Optimal pH increases the sensitivity (staining intensity) as well as the specificity of immunostaining. Acceptable shelf-life of antibodies can also be achieved at optimal pH and dilution in the presence of stabilizing protein (Boenisch, 1999). Unfavorable pH diminishes immunoreactivity because it reduces antibody affinity for the antigen. The role of pH in the interaction between the antibody and the antigen in immunohistological processing is explained below. Antibodies are attracted to the epitopes of most glycoproteins and polypeptides initially through electrostatic charges and subsequently through van der Waals and hydrophobic interactions (Boenisch, 1999). In immune reactions, the isoelectric point (pI) of both antigens and antibodies is therefore of importance. The pI of polyclonal IgGs ranges from 6.0–9.5. Monoclonal antibodies of at least this class possess an equally wide range of individual pI values. If the pH of the diluent and/or solute is used in the same range, the result will be changes in both electrostatic charge and conformation of at least some monoclonal antibodies and possibly of some reactive epitopes. Antibody configuration controls spatial complementarity. All these changes contribute to variable attraction between the antibody and the antigen. As stated above, the pH of the diluent affects the electrostatic charge of monoclonal antibodies and thus the interactions between the antibody and the reactive epitope. Consequently, the optimal operational pH of the monoclonal antibody is determined by the electrostatic charge of the paratope and that of the epitope. Most effective initial attraction between the paratope and the epitope occurs at the pI intermediate to the antigen and that of the antibody. For most antibodies, but not all, this pH is mildly acidic (6.0). An increasingly higher pH of the diluent will decrease the net positive charge of most monoclonal antibodies, resulting in their reduced attraction to negatively charged target epitopes. Higher pH values will also increase the hydrophobicity of antibodies, lessening the interaction between the antigen and the antibody because of the decreased penetration by the latter. It is suggested that new monoclonal antibodies be tested in several dilutions higher than those recommended by the vendor, using 0.05 M Tris buffer (pH 6.0 and 8.6) (Boenisch, 1999). The highest dilution and the pH at which maximal staining occurs should be determined for routine use of the new antibody. This approach frequently allows for the use of antibody dilutions much higher than those recommended by the supplier.

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Note that the use of higher concentrations of monoclonal antibodies does not improve weak staining in immunohistochemistry because of paucity of or masked antigens. Although PBS is commonly used as the antibody diluent, this solution has certain disadvantages. Sodium ions in PBS tend to shield negatively charged epitopes, thereby diminishing the attraction of positively charged reactive sites on the antibody, especially at an alkaline pH. Phosphate ions, on the other hand, promote hydrophobicity. Therefore, the use of PBS and phosphate buffer for diluting the antibodies is not desirable. Accumulated evidence indicates that the most suitable diluent for both monoclonal and polyclonal antibodies is 0.05–0.1 M Tris buffer (pH 6.0) (Boenisch, 2001). The advantage of this pH becomes clear when considering that most antigens and monoclonal antibodies possess opposite surface charges at pH 6.0. Thus, to achieve optimal immunoreactivity, the pH of the environment should be intermediate between the pH of the antigen and that of the monoclonal antibody. Note that any change in the composition and pH of the diluent affects the performance of both the antibody and the antigen. In addition to the pH, the osmolarity of the buffer used to dilute the primary antibody tends to influence the immunoreactivity of monoclonal antibodies. The changes in the molarity of Tris buffer used for diluting monoclonal antibodies are expected to result in changes in the immunoreactivity of antibodies. It has been reported that the higher the concentration of cations (e.g., ) in the buffer or the higher the pH in their presence, the less the immunoreactivity of the monoclonal antibodies (Boenisch, 1999). However, polyclonal antibodies may not show such an adverse effect.

STORAGE OF PARAFFIN-EMBEDDED TISSUES Antigenicity is preserved much better in paraffin-embedded tissue blocks during storage than on the paraffin sections. However, an agreement is lacking regarding the loss of antigenicity due to storage of formalin-fixed and paraffin-embedded tissues. Even similar tissues processed in a similar fashion and stored for the same period of time in different laboratories may show differences in the degree of immunoreactivity. Also, the use of the same antibody when used in different tissues stored for the same duration may yield different degrees of immunohistochemical staining. This predicament is exemplified by the androgen receptor. Androgen receptor activity has been reported to be preserved in the sections of formalin-fixed, paraffin-embedded human archival benign prostate tissue that was stored for up to 16 years (Janssen et al., 1994). In this study monoclonal antibody F39.4 was used; it was raised against a synthetic peptide (SP61) corresponding to the human androgen receptor amino acid sequence 301–320 of the N-terminal domain. In contrast, it has been demonstrated that a significant and persistent decrease in the androgen receptor immunoreactivity occurred in prostatic adenocarcinomas when they were stored for 2 years (Dash et al., 1998). The antibody used was F39.4.1 (BioGenex, San Ramon, CA). Such an immunoreactivity decreased to near zero after 4 years. This decrease begins slowly, followed by more rapid decline, and finally again slows down. In this study, antigen retrieval with microwave heating did not negate the adverse effects of the storage of tissue blocks. Methyl green was used for identifying the tissue background and highlighting the nuclei. Image acquisition and analysis were performed with a CAS 200 image

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analyzer (Becton Dickenson Cell Analysis Systems, Mountain View, CA). Antigen retrieval in both of these studies was carried out using microwave heating. If the preservation of androgen receptor antigenicity is a problem in a formalin-fixed archived tissue, one way to circumvent this problem is to use archived fresh-frozen tissues. On the basis of their study, Dash et al. (1998) have suggested that the use of this antibody for retrospective studies does not correlate androgen receptor status with prognosis or therapeutic response. A decrease in immunoreactivity of p53 antigen in stored paraffin sections is discussed on page 85.

STORAGE OF TISSUE SLIDES Sections of formalin-fixed and paraffin-embedded tissues are commonly used to detect antigens of diagnostic, therapeutic, or prognostic importance in patients with many types of cancers. Therefore, the need for accuracy in the detection of antigen is obvious. Although sections of these tissues are mounted on glass slides usually 1 or 2 days before staining, in some cases paraffin blocks are no longer available. Consequently, immunohistochemistry must be performed on unstained slides that have been prepared some time ago and stored. Because antigen alterations occur on unstained, stored paraffin sections, factors responsible for the alterations need to be understood in order to increase the reliability and quality of immunohistochemical studies (Hayat, 2000a). Most of the technical factors that positively or negatively affect antigen detections are discussed elsewhere in this volume. The following discussion is limited to the clarification of factors influencing the detection of antigens on the stored paraffin sections. Generally, prolonged storage of sections at room temperature results in decreased immunostaining, and thus false-negative staining, which may lead to diagnostic errors and inaccurate prognostic information (Fig. 4.2/Plate 1B-E). In general, antigens that do not require antigen retrieval assistance are less adversely affected during storage than those requiring antigen retrieval with microwave heating or enzyme digestion. Membranous antigens seem to be more adversely influenced by storage of sections than are those located in the cytosol and the nucleus. It is emphasized that antigens affected during storage of sections show decreasing staining with increasing storage temperatures because the stability of most antigens remains intact during storage at 4°C. Decreased immunoreactivity caused by section storage can be compensated for in most cases by using the optimal antigen retrieval method. Different adhesives, such as gelatin and poly-L-lysine, used to ensure section adhesion to the slide do not influence antigen preservation during storage (van den Broek and van de Vijver, 2000). Admittedly, the effect of storage of paraffin-embedded tissue sections on the extent and intensity of immunostaining is controversial. A number of contradictory reports have been published, especially regarding the effects of the type of fixative, duration of fixation with formalin and other fixatives, and temperature of storage (Leong and Gilham, 1989b; Miettinen, 1989; Malmström et al., 1992; Hendricks and Wilkinson, 1994; Bromley et al., 1994; Prioleau and Schnitt, 1995; Kato et al., 1995; Jacobs et al., 1996; McDermott et al., 1997; Shin et al., 1997; Songun et al., 1997; Bertheau et al., 1998; Grabau et al., 1998; Dwork et al., 1998). Recently, excellent, detailed studies of this problem have been carried

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out by Wester et al. (2000) and van den Broek and van de Vijver (2000). These studies and my personal experience are reviewed below. Only a few studies report that prolonged storage of sections does not adversely affect antigen detectability. For example, according to Williams et al. (1997), long-term (6 months at room temperature) storage of sections of tonsil tissue had no effect on the reactivity of the five antibodies tested. In contrast, a vast number of other studies demonstrate decreased staining of stored sections, especially when they are stored at room temperature. It has been demonstrated, for example, that antigens such as p53 and Ki-67 (lung and breast carcinoma) show lower staining after storage for 3 years at room temperature than sections stored for the same period of time at 4°C or –80°C (Grabau et al., 1998). Nuclear estrogen receptor in breast carcinoma also shows higher reactivity when deparaffinized sections are stored for up to 4 weeks in 10% sucrose in PBS at 4°C than that shown by sections stored at room temperature for the same duration (Bromley et al., 1994). This increased staining could be the effect of cold temperature and PBS in which the sucrose is dissolved. It is also known that dissolved salt solutions unmask epitopes in formalin-fixed and paraffin-embedded tissues. It has also been demonstrated that the staining of p53 in mammalian ductal carcinoma decreased after slides were stored for 2 months at room temperature, but the antigen loss

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was significantly less when slides were stored at 4°C (Jacobs et al., 1996). A gradual loss of staining of Ki-67 was reported in the colon tissue when the slides were stored for 9–21 days at room temperature before staining, although a delay of 5 days did not diminish the staining (Holt et al., 1997). Maintaining cut sections refrigerated and protected from light failed to prevent such a loss of MIB-1 immunoreactivity with time. On the other hand, when sections were stored for as long as 1 month at room temperature before staining of PCNA antigen in the same tissue, no adverse effect was observed on the immunoreactivity of PC10 antibody with this antigen. According to Shin et al. (1997), p53 immunoreactivity was not decreased with storage of slides for as long as 25–48 months at room temperature, provided staining intensity is not the only objective of the study. The percentage of positivity of microwave-enhanced immunoreactivity of p53 stored at room temperature and fresh paraffin sections was not statistically significant. Nevertheless, the staining intensity of heated, stored sections was stronger than that in nonheated, freshly cut sections. This study was carried out using tissue blocks of head and neck squamous cell carcinomas stored for 4–15 years and lung carcinomas stored for 14–25 years. Zinc sulfate (1%) was used as the antigen retrieval fluid and was heated for 3 min in the microwave oven. Similarly, the immunostaining of p53 antigen in sections of colorectal carcinoma stored for 6–14 months at room temperature was excellent after microwave heating (Kato et al., 1995). The aforementioned disagreement is due to the study of p53 antigen in different tissues and/or differences in the details of the methodologies used in different laboratories. There are many reasons for the lack of consensus on this highly complex phenomenon, and they are discussed below. Various studies mentioned above were conducted using different parameters of antigen retrieval methods, including antigen retrieval fluids, pH, heat source, temperature, and duration of treatments for detecting different antigens. The type of fixation and duration of fixation also varied in these studies. Other variants were the type of epitope and antibody and source of antibodies used. The degree of immunostaining of stored paraffin sections may differ, depending on whether monoclonal or polyclonal antibodies are employed. Polyclonal antibodies have affinity for several types of epitopes, resulting in positive staining, which may be nonspecific. An antigen retrieval method unmasks more than one type of epitope on the same section, whether stored or not, and such epitopes have access to the polyclonal antibody. However, a recent study indicates that polyclonal antisera show only slightly better staining than that obtained with monoclonal antibodies (van den Broek and van de Vijver, 2000). It is also possible that loss of immunostaining in stored sections is epitope related instead of related to the antigen as a whole (Henson, 1996). In addition, tissue heterogeneity at different levels was not considered. The quantity and quality of antigen reactivity varies from one tissue block to another. In some of these studies the automated immunostainer was used, whereas in others manual staining was carried out. The automated stainers reduce human procedural errors, and their controlled environment increases the speed and timeliness of results in high-volume laboratories, although their high cost might be prohibitive for small laboratories. Another factor that may affect results is that a collection of stored sections may be heterogenous regarding duration of storage because of the successive addition of new sections. Subjective evaluation or inadequate quantification of the extent and intensity of staining was the common

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denominator in most of these studies. These are the main reasons for the contradictory results reviewed above. Attempts have been made to protect sections from exposure to oxygen during storage by coating them with paraffin (Jacobs et al., 1996; Rittman, 2000). This is accomplished by heating the slide to ~60°C and placing a few drops of molten paraffin on it; a second heated slide is gently drawn across the surface of the first slide to form a thin protective paraffin layer on the sections. In our experience, coating the surface of paraffin sections mounted onto slides with paraffin does not significantly reduce antigen loss after their storage for several weeks or months at room temperature or at 4°C. Xylene-based spray glue has also been used for protecting the stored sections but without success (Wester et al., 2000). Different antigens are affected differently by the storage of sections of even the same formalin-fixed, paraffin-embedded tissue. In other words, the degree of the antigenicity loss due to storage of the unstained slides differs depending on the type and location of the antigen. For example, nuclear steroid receptors tend to be comparatively more sensitive to storage (aging). Comparative studies, using a panel of eight antibodies against Ki-67, prostatic-specific antigen, androgen receptor, epidermal growth factor receptor, and prostatic acid phosphatase, demonstrated that nuclear androgen receptor showed a higher decrease of antigenicity in stored, unstained sections compared with that exhibited by other antigens (Olapade-Olaopa et al., 2001). The loss of antigens due to storage of paraffin sections is not a serious problem in many clinical immunohistochemistry laboratories that perform immunostaining within hours or days after paraffin sections have been cut. However, antigen loss may become a problem when slides are stored for months at room temperature as positive controls. Such storage is encountered in some research laboratories where unstained paraffin sections are archived for future use. In any case it is recommended that sections be stained rapidly after they have been cut from the formalin-fixed, paraffin-embedded tissues. It is likely that prolonged storage of sections at room temperature strengthens protein crosslinks, which become less reversible, resulting in diminished antigen retrieval. If immunostaining needs to be postponed, tissue specimens should be stored in paraffin blocks rather than as paraffin sections because antigenicity is better preserved in the former state. The results of a comprehensive study on the effects of fixation, temperature and duration of section storage, and antigen retrieval on the immunostaining of p53 antigen are shown in Fig. 4.3 (Plate 2). Color-based image analysis was used to quantify the extent and intensity of staining. In conclusion, the storage of paraffin sections decreases, to a varying degree, immunoreactivity for most, but not all, antigens. The maximal decrease in immunoreactivity, at least of p53 and Ki-67 antigens, occurs during the first 2 weeks of storage (Wester et al., 2000). The decrease in immunoreactivity is generally inversely related to an increase in storage temperature. Both the extent and intensity of staining tend to be negatively influenced by storing the sections. The decreased immunoreactivity as a result of section storage can be compensated for in most cases by using optimal antigen retrieval procedure. Although longer durations of fixation are accompanied by increased masking of antigens during section storage, this relationship is neither universal nor linear. If paraffin sections must be stored, they should be stored at –20°C, irrespective of the duration of storage.

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SIGNAL AMPLIFICATION Most antigens are not destroyed during fixation with formaldehyde, but are reversibly masked. Methods to unmask them are presented in this volume. However, some antigens are difficult to visualize adequately with routine immunohistological techniques and therefore require signal amplification with an acceptable signal-to-noise ratio. An amplification of immunostaining intensity is especially useful when monoclonal antibodies are used because they bind only a single epitope. Small amounts of antigens in tissue sections can be detected specifically by using signal amplification. Techniques used for increasing the sensitivity or signal amplification are summarized below. A number of strategies have been employed for improving immunohistochemical signals. The immunofluorescence antibody method was developed for specific identification of cells based on their antigen makeup (Coons et al., 1941). Its use is limited because of the need for fresh-frozen sections and inadequate preservation of cell morphology. Also, the fixed ratio of fluorescein to the antibody does not allow amplification of the signal. The peroxidaselabeled antibody method is more compatible with the basic substrates of surgical pathology specimens fixed with formalin and embedded in paraffin (Nakane, 1968). This immunoperoxidase protocol can be amplified by increasing the duration of development. The original immunoenzyme bridge method using enzyme-specific antibody (Mason et al., 1969) has been superseded by an improved technique using a soluble peroxidase antiperoxidase complex (PAP) (Sternberger et al., 1970). These complexes are formed from three peroxidase molecules and two antiperoxidase antibodies and are used as a third layer in the staining method. They are bound to the unconjugated primary antibody (e.g., rabbit antihuman IgG) by a second layer of bridging antibody (e.g., swine antirabbit immunoglobulin), which is applied in excess so that one of its two identical binding sites binds to the primary antibody and the other to the (rabbit) PAP complex. The PAP method is more sensitive than indirect methods using fluorescein or peroxidase-conjugated antisera. Alkaline phosphatase antibodies raised in the mouse can, by the same principle, be used to form alkaline phosphatase anti-alkaline phosphatase (APAAP) complexes. These have uses and advantages similar to those of the PAP complexes. The avidin-biotin methods rely on the marked affinity of the glycoprotein avidin for biotin. Avidin is composed of four subunits which form a tertiary structure possessing four biotin-binding hydrophobic pockets. The oligosaccharide residues in avidin give it some affinity for the tissue components, especially some lectinlike proteins, and result in nonspecific binding. A similar molecule, streptavidin, has some advantages over avidin, as the former lacks oligosaccharide residues and possesses a neutral isoelectric point. The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complexing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotinlabeled antibody. In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracer containing the free avidin binding sites is applied to the biotinylated antibody. As a high

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number of biotin molecules can be attached to a single antibody, a high tracer-to-antibody ratio can be achieved. This property yields high sensitivity and allows the use of an increased dilution of the primary antibody. The most recently developed and refined signal amplification method is termed catalyzed reporter deposition (CARD), which is discussed below. The historical development of signal amplification methods has been presented by Elias (1999).

Tyramine Amplification Method The tyramine amplification method is based on the characteristic ability of tyramine to become chemically adhesive following oxidation/radicalization (Gross and Sizer, 1959). Oxidation of the tyramine generates a brown pigment that contains dityramine and more extensively oxidized and polymerized derivatives. Bobrow et al. (1989, 1991, 1992) used tyramine for enhancing ELISA, and Adams (1992) adapted it for immunohistochemistry. Subsequently, Mayer and Bendayan (1997) extended the application of the tyramine signal amplification to immunoelectron microscopy (Fig. 4.4). Van Gijlswijk et al. (1997) used green, red, and blue fluorescent tyramides in immunohistochemistry, immunocytochemistry, and fluorescence in situ hybridization. Other applications of tyramine include Western blotting (Wigle et al., 1993) and in situ hybridization (Kerstens et al., 1995). Tyramine amplification is an important development in advancing the efficiency of immunohistochemistry, and its further applications are expected. The catalyzed reporter deposition (CARD) amplification signal method was initially described by Bobrow et al. (1989). It is based on the deposition of biotinylated tyramine at the location of the probe catalyzed by horseradish peroxidase (HRP). It has been established that the highly reactive intermediates formed during the HRP-tyramide reaction will bind to tyrosine-rich moieties of proteins present in the vicinity of the HRP binding sites. The binding of tyramine to proteins at the site of HRP occurs via the production of free radicals by the oxygen liberated by HRP. In other words, HRP reacts with and the phenolic moieties of tyramine to produce a quinonelike structure bearing a radical on the C2 group. Because this reaction is very short lived, deposition occurs only in the location at or in immediate proximity to where it is generated. The biotin conjugated to the bound tyramine is subsequently used for the attachment of avidin, which is conjugated to HRP. This HRP is then used to catalyze the brown color reaction. This method allows highresolution detection of primary antisera because the tyramide complex precipitates only at the site of reaction. Toda et al. (1999) have compared the immunostaining using conventional avidin-biotin complex (ABC) with tyramide signal amplification-avidin-biotin complex (TSA-ABC); relatively distinct staining was apparent in the latter technique. As the TSA-ABC protocol dramatically improves the signal intensity by the peroxidasecatalyzed deposition of biotinylated tyramide, blocking of endogenous peroxidase is required. To ensure quenching of the residual peroxidase, the use of a higher concentration and duration of treatment with is recommended. The amplification power of TSA-ABC can be enhanced by using several subsequent cycles of incubation or by extending the duration of incubation, for example, up to 30 min at 37°C. Longer incubations may result in background noise (nonspecific staining). Also, the number of cycles possible before the background noise level becomes unacceptable is

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limited to two or three. It should be noted that because the tyramide deposition reaction is rapid, small differences in amplification duration may lead to variations in the final signal intensities. Furthermore, because this reaction amplifies both specific and nonspecific immunohistochemical signals, it is essential that appropriate positive and negative controls be used to achieve correct interpretation of staining. Also, because TSA-ABC tends to enhance the background noise along with the signal, the procedure must be optimized to ensure low nonspecific binding. All tyramide conjugates yield approximately the same

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results, indicating that signal amplification is independent of the tyramide conjugate used (Speel et al., 1998). Numerous biotin conjugated tyramides can be detected with avidinconjugate (Totos et al., 1997). If biotin reaction fails, the primary reason is the age of the biotin solution. Because the shelf-life of newly synthesized biotin is not known, one should at least be aware of the expiration date of the reagent. Different authors and commercial suppliers have assigned different names to signal amplification using tyramine. For example, tyramine signal amplification (TSA) system and the catalyzed signal amplification (CSA) system are commercially available from DuPont NEN Life Science Products, Boston, MA, and DAKO Corporation, Carpinteria, CA, respectively. In addition, the terms CARD (catalyzed reporter deposition) (Bobrow et al., 1989), TA (tyramide amplification) (Shindler and Roth, 1996), and ImmunoMax (Merz et al., 1995) have been used for the tyramine amplification technique. The use of different names for almost identical procedures has resulted in confusion. To standardize the terminology, the neutral abbreviation, tyramide amplification technique (TAT) should be accepted (Von Wasielewski et al., 1997). One of the variations of the tyramide amplification technique is termed ImmunoMax (Merz et al., 1995). In this approach the biotinylated tyramine enhancement is combined with an antigen retrieval method such as microwave heating, enzyme (proteinase K) digestion, or exposure to a detergent (guanidine hydrochloride). This method is effective in detecting some previously unreactive, inadequately reactive, or partly demasked antigens in the formaldehyde-fixed and paraffin-embedded tissues. It has been claimed that this technique allows as much as 10,000-fold dilution of the primary antibody and 100 to 1,000-fold increase in sensitivity compared to those used with the conventional ABC method (Merz et al., 1995). However, the sensitivity increase in the range of 5- to 50-fold is more feasible (Speel et al., 1999). Preparation of Biotinylated Tyramine

One hundred milligrams of sulfosuccinimidyl-6- (biotinimide) hexanoate (NHS-LCbiotin) (Pierce, Rockford, IL) is dissolved in 40 ml of 50 mM borate buffer (pH 8.0). To this solution is added 30 mg of tyramine hydrochloride (Sigma Chemical Company, St. Louis, MO). The solution is stirred overnight at room temperature and filtered ( filter). The final biotinylated tyramine concentration is Before application, the solution is diluted 1:160 in Tris-HCl buffer (pH 7.6) containing 0.03%

Rolling Circle Amplification Although immunohistochemistry is a versatile and powerful tool for various molecular and cellular analyses, especially for antigen detection, it has a few limitations, such as lack of standardization and difficulty in visualization of antigens present at low concentrations. In numerous instances important biological markers for cancer, infectious disease, and biochemical processes are present at too low a concentration in tissues or body fluids to be detected by conventional methods. The difficulty of detection of low concentrations of antigens can be minimized by antigen retrieval using heating or enzymatic digestion.

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This problem can also be lessened by using stronger fluorochromes and chemiluminescent substrates for use in ELISAs, immunofluorescence-based staining, and immunoblotting. Detection of low concentrations of antigens can also be achieved by increasing the signal without raising the level of nonspecific background staining. Signal amplification, for example, can be achieved by successive steps of enzymatic reactions. Biotinyl tyramide is commonly used to increase the signal of low abundance targets that are otherwise undetectable by conventional techniques. However, tyramide-based amplification may increase background noise because of multiple steps of signal amplification (discussed in this chapter). Therefore, molecular tissue pathology requires techniques of greater sensitivity and specificity. One of such techniques to refine the examination of cell components is rolling circle amplification (RCA) discussed below (Lizardi et al., 1998). Rolling circle amplification is essentially a surface-anchored DNA replication that can be used to visualize single molecular recognition events. It is an isothermal nucleic acid amplification protocol that differs in several aspects from the polymerase chain reaction (PCR) and other nucleic acid amplification methods. The RCA can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. It has sufficient sensitivity to detect individual oligonucleotide hybridization events and single antigen-antibody complexes (Schweitzer et al., 2000). The linear mode of RCA can generate signal amplification during a brief enzymatic reaction. Another advantage of linear RCA is that the product of amplification remains connected to the target molecule. Signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules (Zhong et al., 2001). This protocol has been used for visualizing target DNA sequences as small as 50 nts long in peripheral blood lymphocytes or in stretched DNA fibers (Zhong et al., 2001).

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Chapter 5

Problems in Antigen Retrieval

LACK OF IMMUNOSTAINING The failure of antibodies to immunostain tissues or cells does not necessarily reflect the absence of epitopes. Lack of, or reduced, immunostaining can be attributed to multiple factors. The most common factor is the inability of the antibody to reach and recognize the epitope under the preparatory conditions used, including fixation, dehydration, embedding, deparaffinization, rehydration, and incubation. The inaccessibility of the epitope to the antibody may be due to the formation of large, compact protein complexes as a result of crosslinking by formaldehyde. These complexes create a barrier to antibody penetration. This aspect of immunostaining failure is elaborated upon later. It is also possible that the antigen molecule is folded and thus hides, the epitope, especially from monoclonal antibodies. Apparently, better immunostaining depends on improving antibody access to, and recognition of, the epitope. It is well established that many types of antigen molecules are altered by dehydration solvents and other reagents. Lack of immunostaining may also be due to excessively diluted antibody, to loss of antibody owing to degradation by bacteria or fungi, or to antibody aggregation due to repeated freezing and thawing. Finally, a monoclonal antibody will not recognize an epitope in vivo if the former is raised against a denatured antigen. This is also true for polyclonal antibodies when the recombinantly produced antigen becomes denatured during isolation and purification (Binder et al., 1996). Fixation with aldehydes plays a key role in the two above-mentioned events: antibody access to and recognition of the epitope. Tissues and cell cultures are usually fixed with an aldehyde prior to immunostaining. Fixation has the advantage of anchoring in situ antigens, as aldehydes are powerful protein crosslinking agents. They crosslink proteins and glycoproteins through reversible and irreversible alterations in the molecular conformation of proteins, including antigens (epitopes). If the change in conformation is strongly irreversible, the antibody will have difficulty recognizing the altered epitope, especially aldehyde-sensitive antigens. This problem is especially acute when specimens are fixed with glutaraldehyde. This effect of aldehydes on epitopes and their surrounding proteins is called epitope masking. Briefly, epitope unmasking can be accomplished by weakening or breaking down the protein crosslinking introduced during aldehyde fixation and allowing the epitope to be exposed to the antibody, provided the latter has access to the former. Thus, two simultaneous events (unmasking of epitope and access of antibody to epitope) 95

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must take place to achieve immunostaining. Detailed methods of unmasking or retrieval of epitopes after fixation with an aldehyde are discussed later. Note that there is no single, ideal pretreatment for retrieving all types of epitopes; the optimal treatment for a given epitope may not be effective for another type of epitope. This is particularly true when considering the pH of the epitope retrieval solution and the duration of pretreatment. These and other pretreatment conditions are explained later. Note also that less than optimal processing conditions for a given epitope may also result in background immunostaining.

BACKGROUND STAINING Background staining is one of the common problems in immunohistochemistry, and it has a number of causes, which are discussed below. One cause is protein hydrophobicity, which can occur between different protein molecules. Fixation with aldehydes renders proteins more hydrophobic as a result of crosslinking between reactive amino acids. The crosslinkages are both intramolecular and intermolecular (Hayat, 2000a). The extent of hydrophobic crosslinking depends on the duration, temperature, and pH of fixation. Because changes in these factors result in variable hydrophobicity, owing to variable protein crosslinking, once an optimal fixation is determined, it must be maintained and controlled. According to Boenisch (2001), excessive background staining resulting from overfixation with formalin can be remedied by postfixation with Bouin’s or B5 fixative. It should be noted that the greater the proximity of the pH of the antibody diluent and the isoelectric point (pI) of the antibody, the stronger the hydrophobic interaction. In contrast, the lower the ionic strength of the diluent, the weaker the strength of hydrophobic attraction. Hydrophobic interactions can also be reduced by adding a detergent, such as Tween 20, to the antibody diluent. The best approach to significantly reduce background staining due to hydrophobic interaction is to use a blocking protein immediately before or also during the application of the primary antibody. The blocking protein must be of the type that competes effectively with IgG for hydrophobic binding sites in the tissue. Also, the blocking protein should be identical to that present in the secondary link or labeled antibody, but not that in the primary antibody, in order to avoid nonspecific binding of the secondary antibody. To fulfill these requirements, 1% bovine serum albumin (BSA) is added to the primary antibody diluent. Nonfat dry milk or casein can be used in place of BSA. The cross-reactivity of antibodies can also cause background staining. This problem arises when the epitope under study is shared among different proteins in the target tissue. Use of polyclonal antibodies can result in nonspecific cross-reactivity with similar or dissimilar epitopes on different antigens. Because an unabsorbed antiserum tends to increase this problem, it should be subjected to careful affinity absorption. Use of antibodies from hyperimmunized animals will also help. Careful screening of clones in the case of monoclonal antibodies will eliminate this type of background staining. Antibody crossreactivity has been discussed in more detail in Chapter 2. The presence of even small amounts of natural antibodies in the serum may also produce nonspecific staining. These antibodies result from prior environmental antigenic stimulation. In fact, such antibodies may increase in the titer during immunization of the

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animal with adjuvants. Although these antibodies are difficult to remove, their net effect can be almost eliminated by using the antiserum at a sufficiently high dilution or by reducing the duration of incubation. Nonspecific staining may also result from contaminating antibodies produced by the host’s immune system as it reacts to isolated antigens used for immunization (Boenisch, 2001). Isolated antigens are rarely pure. If these antibodies are a problem, the antiserum should be subjected to affinity absorption. Fortunately, such antibodies are present in a very low concentration and may not cause troublesome background staining. Use of hightitered antisera at sufficiently high dilutions would eliminate this problem. Natural and contaminating antibodies do not cause any problem when using monoclonal antibodies. Nonspecific staining can be caused by Fc receptor glycoproteins present on the cell membrane. This problem is more relevant to frozen sections and smears than to tissues fixed with formaldehyde. The problem can be avoided by using fragments instead of whole IgG molecules (Boenisch, 2001). Complement-mediated binding may also cause background staining in frozen sections when whole antisera is used; however, this problem is not very common. Antigen diffusion can cause specific background staining. This problem arises when the target antigen is displaced from its site of synthesis or storage. Delayed fixation and/or incomplete fixation with formaldehyde tend to cause this problem. Optimal fixation with this monaldehyde anchors the antigens at their site of synthesis. Mechanical injury to the tissue or drying of the tissue prior to fixation may result in diffuse background staining. Necrotic areas due to autolysis of the tissue tend to stain with almost all staining reagents. Antigen retrieval with prolonged enzyme digestion often disrupts cell architecture, resulting in the displacement of target antigens from their site of greater density; the net effect is increased background staining. Background staining also results from the presence of endogenous peroxidase in the formalin-fixed tissues. This artifact can be avoided by treating the tissue sections with 3% hydrogen peroxide in water for 4–9 min at room temperature; methanolic hydrogen peroxide is not recommended. Blocking of the endogenous peroxidase activity is especially desirable with cell preparations and frozen sections (Boenisch, 2001). Endogenous biotin, distributed in a wide variety of tissues, may also cause background staining with biotin-based immunohistochemical techniques. This biotin is especially abundant in liver, whereas it is poor in the central nervous system and adipose tissue. Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but is also present in sections of paraffin-embedded tissues. This problem is largely eliminated by using streptavidin-based methods or by sequential treatment of sections (prior to staining) with 0.01–0.1% avidin followed by 0.001–0.01% biotin for 10–20 min each. The biotin problem is discussed in more detail later in this chapter. Other causes of diffuse background staining include the presence of residual embedding medium and bacterial or yeast contamination in the water bath. The presence of undissolved chromogen granules on occasion may create the problem of nonspecific staining. Excessive counterstaining with reagents, such as hematoxylin and eosin, may compromise specific staining. Finally, a few published reports indicate that antigen retrieval at extremely high temperatures may result in nonspecific staining. Baas et al. (1996), for example, have reported false-positive results at a very high antigen retrieval temperature using monoclonal

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antibody D07 against p53 antigen. They carried out antigen retrieval at 96°C for 30 min in the Target Unmasking Fluid (TUF) containing 35% urea in a microwave oven. This combined treatment is unusually excessive and is not used routinely.

PROBLEM OF ENDOGENOUS BIOTIN The presence of endogenous biotin is a known potential source of nonspecific staining in immunohistochemical methods based on the avidin-biotin system (Fig. 5.1/Plate 3A and B). Biotin is a water-soluble monocarboxylic acid (a vitamin) of molecular weight 244 Da in living cells. This vitamin functions as a prosthetic group for carboxylase enzymes used in fatty acid biosynthesis and gluconeogenesis. It is widely distributed in many tissue types, including liver, kidney, breast, pancreas, salivary glands, skeletal and cardiac muscles, adipose tissue, and a variety of neoplasms (e.g., salivary gland neoplasm [Lu et al., 2000]). Biotin has been demonstrated immunohistochemically, for example, in human thyroid, parathyroid, adrenal, salivary, mammary, and prostate glands (Green et al., 1992). The presence of cytoplasmic endogenous biotin has also been demonstrated in thyroid papillary carcinoma (Kashima et al., 1997). This vitamin is found both in the cytoplasm and in the mitochondria. The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) method are more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemical techniques. The strong noncovalent attraction between biotin and avidin or streptavidin is exploited in many histochemical, immunohistochemical, and in situ hybridization

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procedures discussed in this volume. As an example, avidin-peroxidase and streptavidinperoxidase conjugates and antibiotin antibodies are routinely used for labeling biotinylated antibodies that bind to antigens. In addition, antibiotin antibodies are employed in multistep protocols to enhance the sensitivity of immunohistochemical and in situ hybridization methods (McQuaid and Allan, 1992). However, the avidin-biotin and the streptavidin-biotin detection systems in some cases can lead to false-positive immunostaining. The avidin conjugates employed in these biotinavidin methods bind to the endogenous biotin, thereby resulting in artifactual staining. Such spurious staining has been documented in the above-mentioned tissues as well as in such disparate tissues as gestational endometrium and ovarian lipid cell tumors (Seidman et al., 1995). Another example of false-positive immunostaining is the purported presence of inhibin in hepatocellular carcinomas (McCluggage et al., 1997). However, a recent study has demonstrated that when endogenous biotin is blocked, immunostaining of inhibin in hepatocellular carcinomas and hepatocytes is absent (lezzoni et al., 1999). By blocking endogenous biotin, highly specific staining of endothelium using CD-34 as an antibody without nonspecific tubular staining has been achieved in the kidney tissue (Rodriguez-Soto et al., 1997). The frequency of such erroneous staining is likely to increase as the sensitivity of the protocols for labeling biotin improves (i.e., biotin amplification techniques) (Adams, 1992). It is thought that the problem of endogenous biotin staining is more serious with some antibiotin antibodies than with streptavidin conjugates (Cooper et al., 1997). The problem depends on the affinity/sensitivity of the antibody used. Also, the problem becomes more prevalent when tissues are pretreated with detergents or digestive enzymes for antigen retrieval (Satoh et al., 1992). Moreover, the intensity of this artifact is enhanced by heat-induced antigen retrieval methods. This nonspecific staining is also observed in in situ hybridization with biotinylated probes (Kashima et al., 1997). The presence of this artifact poses a distinct risk of its being interpreted as positive staining, as the artifact can be intense and may be precisely located in the cells of interest with a clean background. It is known that liver and kidney can retain high amounts of retrievable biotin-avidin activity in neoplasms. The need for adequate controls or biotinblocking procedures is obvious when histochemical or immunohistochemical procedures are used. Negative controls facilitate identification of such nonspecific staining. An alternative to the avidin-biotin technology, the EnVision™+System (Dako) detection method, is recommended for universal use in diagnostic and research studies. It is based on enhanced polymer methodology. In comparison with APAAP, PAP, ChemMate™, CSA, LABC, and SABC methods, the En Vision™+System yields optimal detection (Sabattini et al., 1998). Its sensitivity is at least as good as that of Strept ABC techniques, and its use completely eliminates the problem of endogenous biotin. Another more recently introduced method to prevent endogenous biotin staining consists of using a nonbiotin amplification (NBA) detection system (Zymed, San Francisco, CA) (Shi et al., 2000b). This method is as effective as the conventional technique using the Lab-SA kit (Histostain-Plus kit, Zymed) but avoids nonspecific biotin staining. The NBA kit is composed of an FITC-labeled secondary antibody and a horseradish peroxidase– conjugated anti-FITC antibody. Figure 5.2 (Plate 1F and G) shows HER-2 staining in the infiltrating ductal carcinoma cells of breast using the Lab-SA kit or the NBA kit. If either of the two methods mentioned above is not used, the following procedure can be employed to avoid endogenous biotin staining.

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Endogenous biotin-avidin activity can be blocked by treating the sections of formalinfixed and paraffin-embedded tissues with avidin solution alone or followed by biotin solution. These treatments are carried out after heating for antigen retrieval, and they do not interfere with subsequent immunoperoxidase staining. The concentration of the avidin is critical in blocking endogenous biotin. A cost-effective alternative to commercially available avidin, which is rather expensive, is dilute egg white (Miller and Kubier, 1997). Avidin solution can be prepared by mixing two egg whites in 200 ml of distilled water. Skim milk should not be used as a substitute for commercially available biotin (R.T. Miller, personal communication). Use of 0.2% biotin in PBS is recommended. The specificity of this method requires the absence of both avidin-binding sites and peroxidase activity in the tissue sections. Immunoperoxidase staining of endogenous biotin in frozen sections can be eliminated by their treatment with 1% hydrogen peroxide in methanol (Cooper et al., 1997). Such treatment should be applied after application of primary antibody.

Procedure (lezzoni et al., 1999) Sections ( thick) of formalin-fixed and paraffin-embedded tissues are placed on slides, dried overnight at 37°C, deparaffinized with xylene, and rehydrated with descending concentrations of ethanol. They are treated with 3% hydrogen peroxide in methanol for 5– 10 min and then rinsed with PBS. The slides are immersed in 10 mM citrate buffer (pH 6.0), and heated for 2 min at 100% power, followed by 8 min at 80% power. After being rinsed in PBS, the sections are incubated in an appropriate primary antibody. They are rinsed in PBS and then treated for 4–8 min with egg white avidin solution (2.5 g of egg white avidin in 0.1 mM PBS) (Ventana Medical Systems). Avidin binds to endogenous biotin. Following rinsing in PBS, the sections are treated with free biotin solution (2.5 mg in 0.1 mM PBS) for 4 min to saturate the remaining binding sites of the egg white avidin. The sections are thoroughly rinsed with tromethamine-based buffer or PBS, followed by the following sequential immunostaining protocol: biotinylated secondary antibody,

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avidin-strep+avidin-horseradish peroxidase conjugate, DAB-hydrogen peroxide, copper enhancer, and hematoxylin. The slides are dipped in lithium carbonate, and cover-slipped.

MIRROR IMAGE COMPLEMENTARY ANTIBODIES The conventional immunodetection system may contribute to poor staining specificity because of unwanted interactions between the immunoreagents and the endogenous tissue components. Avidin-biotin-based systems, for example, may cause unwanted staining by reacting with endogenous biotin. Another example is the presence of the endogenous enzyme, which tends to cause nonspecific chromogen precipitation. To avoid some of these problems, Mangham and Isaacson (1999) introduced a novel and sensitive peroxidasebased immunohistochemical detection method that employs mutually attractive, mirror image complementary antibodies (MICA). Such antibodies consist of two polyclonal antibodies raised in different species that are mutually attractive, i.e., they are raised against each other’s immunoglobulin species. Thus, each antibody is both an antigen and an antibody with respect to the other. Compared with the ABC technique, the MICA method allows up to 200-fold dilution of the primary antibodies with equivalent or superior immunostaining and shorter durations of incubation. Other advantages of the MICA method are that it is avidin-free and thus avoids nonspecific staining due to endogenous biotin, and yields ~64-fold increase in sensitivity (as judged by dot-blot) compared with that of the ABC technique. The improved sensitivity of the MICA protocol is thought to be due to increased stability of the complexes produced and possibly to antigen bridging. A minor limitation is the longer duration required to complete this method.

Procedure Freshly paraffin-embedded or archival paraffin-embedded tissues can be used (Mangham and Isaacson, 1999). Sections ( thick) are deparaffininzed in xylene and rehydrated in descending concentrations of ethanol. Antigen retrieval is carried out by heating the sections in 1 mM EDTA (pH 8.0) in a pressure cooker at full pressure for 2 min. The sections are allowed to cool and are then transferred to TBS (pH 7.4). They are treated with 3% in methanol for 15 min to block endogenous peroxidase activity and washed in TBS/Tween ( Tween/1 ml TBS). The sections are incubated in the primary antibody (diluted in TBS) for 1 hr and then washed in TBS/Tween. They are incubated in 1:30 diluted link antibody (sheep antimouse Ig or sheep antirabbit Ig) (the MICA target antibody) for 20 min, and then washed in TBS/Tween. The sections are incubated in 1:30 diluted MICA antibody No. 1 (peroxidaseconjugated donkey antisheep Ig) for 20 min and washed with TBS/Tween. They are incubated in 1:30 diluted MICA antibody No. 2 (sheep antidonkey Ig) for 20 min and washed in TBS/Tween. This is followed by incubation with 1:30 diluted MICA antibody No. 1 (peroxidase-conjugated donkey antisheep Ig) for 20 min and washed in TBS/Tween. The sections are exposed to 1 mg/ml DAB-0.02% in TBS for 6 min and washed in TBS. They are dehydrated in ethanol, counterstained with hematoxylin, and cover-slipped.

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All the reagents are commercially available in kit form (polyMICA: Binding Site Ltd., Birmingham, UK).

FIXATION OF FROZEN TISSUES Occasionally, tissues frozen for intraoperative consultation are processed for immunostaining. This situation arises, for example, when lesional tissue or atypical cells that are seen in a frozen section require confirmation. Some antigens show decreased or negative staining in tissues that are frozen before fixation with formalin, whereas the staining of some other antigens remains unchanged. For example, the staining of S-100, HMB45, synaptophysin, and neuron-specific enolase was negative in frozen tissues that were subsequently fixed with formalin, but the staining of these antigens was positive in freshformalin-fixed tissues (Edgerton et al., 2000). The staining of chromogranin was decreased in frozen-fixed tissues. In contrast, the staining of cytokeratins remained unchanged in frozen-fixed tissues. This and other evidence indicates that sections of frozen tissue that have been subsequently fixed with formalin may show false-negative staining. Although the exact mechanism responsible for the above-mentioned false-negative staining is not known, it is likely that cell membranes are disrupted when frozen tissue is thawed during fixation. Such a disruption does not occur when fresh tissue is fixed. Thus, damaged membranes would facilitate antigen diffusion out of the nucleus and/or the cell. This problem is especially serious for antigens in neural tissues (Edgerton et al., 2000); therefore, caution is warranted in interpreting immunohistochemical results of tissues that are fixed preceded by freezing. It is recommended that when surgeons freeze the tissue specimen, they should also fix freshly cut specimens of the same tissue for comparative study. Use of frozen sections without postfixation also has limitations in certain cases. The following example testifies to the drawback of using such sections for diagnostic purposes. Although intraoperative frozen-section evaluation for the surgical treatment of Hirschsprung’s disease is a common practice, a high rate of incorrect diagnosis of this disease has been reported using frozen sections (Maia, 2000). When surgical pathologists use primary resection without prior colostomy or frozen sections as the initial diagnostic test, the results of an incorrect frozen section could be disastrous. Because the concordance rate for frozen-section diagnosis on initial pathological specimens is low (67%), establishing an initial diagnosis of Hirschsprung’s disease on frozen sections is not recommended. Furthermore, the introduction of artifacts tends to make interpretation of already subtle histological findings untenable. It is therefore recommended that well-prepared permanent sections be used to establish the absence of ganglion cells in a rectal biopsy for the presence of this disease. It is known that pathological diagnosis of Hirschsprung’s disease is established by demonstrating the absence of ganglion cells in the colonic neural plexuses.

HOT SPOTS (AREAS) IN MICROWAVE OVEN Microwaves consist of electric and magnetic fields, and they propagate in space. Electric fields are primarily responsible for physical effects on the tissue. The energy distribution and thus the speed of absorbing energy and warming up vary topographically. In

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other words, the spatial energy distribution in a microwave oven is unequal. A region in the oven with a high intensity of electromagnetic fields is known as a hot spot or, more correctly, a hot region (~1 cm). Hot areas are not located at fixed coordinates in the oven and are influenced by the load placed in the oven. Loads of various sizes and shapes lead to different heating patterns. By varying the position of the load within the oven during irradiation, reproducible results can be obtained. The problem of hot areas can be solved by providing a microwave transparent rotating platform for the load during irradiation and by placing an extra load to serve as a heat sink; a jar containing 100 ml to 1 liter tap water or antigen retrieval solution suffices. To obtain reproducible results the same type of jar should be placed in the same location in the oven. For detailed theoretical and practical considerations of hot areas, the reader is referred to Kok et al. (1993).

PROBLEM OF ANTIGEN RETRIEVAL STANDARDIZATION Lack of reproducibility of results in epitope retrieval immunohistochemistry is a serious problem unacceptable in diagnostic immunopathology. Variable intra- and interlaboratory results are a common phenomenon. Lambkin et al. (1998) have assessed immunohistochemical results of estrogen receptor obtained from 16 Irish histopathology laboratories. They confirmed that although the majority of participants achieved acceptable immunopositive staining in the supplied sections, variations in the intensity of immunostaining, focal staining, and nuclear staining were observed. Battifora (1998) has discussed the necessity of minimizing at least intralaboratory variability of results, which is quite feasible. Both Lambkin et al. (1998) and Battifora (1998) have suggested means by which some degree of reproducibility of results can be obtained. A large number of factors influence the final results of immunostaining: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Fresh or archival tissue specimens Type of fixative and its pH and concentration Duration of fixation, especially for formalin-sensitive epitopes Duration of tissue storage in formalin Embedding procedures Type of retrieval fluid and its pH Type of microwave oven and heating parameters, other heating sources, or other epitope retrieval treatments such as enzyme digestion and ultrasound Monoclonal or polyclonal antibodies Tested or new antibodies obtained from the same clone or not Source of antibodies; even similar monoclonal antibodies obtained from different sources may show differential affinity for the epitope Incubation times and temperatures Variable staining methods; automated immunostainers or manual staining Differences in the interpretation of results

Considering the processing variables enumerated above and below, achieving complete standardization of immunoreactions is extremely difficult. Standardization of fixation is difficult due to the large number of variables, including the volume and concentration of

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the fixative, temperature and duration of fixation, size and composition of the tissue, and the amount of free blood. Also, irrespective of the size of the tissue block, the fixation is variable within the block. The same duration of fixation with formaldehyde may or may not result in identical fixation, for each tissue block responds uniquely to the fixative. Moreover, uniform sections are difficult to obtain, and their thickness is difficult to determine. Many sections are wedge-shaped rather than planoparallel (Rittman, 1998). In addition, the arc of vibration caused by the knife edge as it cleaves the section is sufficient, for example, to glide over or under the surface of some nuclei or cut through the remainder. Consequently, an accurate measurement of the concentration of nuclear antigens is difficult (Allison, 1999). The tendency is to cut the thinnest possible sections to obtain superior resolution that provides distinct images. However, thin sections show excessive compression as well as variation in thickness because compression is usually inversely proportional to section thickness. Another obstacle is the small number of sections that are usually examined in a diagnostic laboratory, limiting the production of reliable average or quantitative data.

TEST BATTERY A standardized method for retrieving a given epitope in a particular tissue can be developed by using the test battery approach (Shi et al., 1996a). This is a convenient and rapid means to optimize three important factors (pH, temperature, and duration of heating) responsible for the immunostaining of a given epitope-antibody combination. The optimal protocol lessens false-negative immunostaining. The need for this approach arises when optimal conditions for retrieving an epitope are not known. It is known that the retrieval of different epitopes requires specific retrieval conditions. These conditions primarily consist of the pH and the temperature of the epitope retrieval fluid in the microwave oven and the duration of heating. Other factors influencing the final results of immunostaining are not included in this test. The following three levels of heating durations and three pH levels of the epitope retrieval fluid (sodium citrate buffer and Tris-HCl buffer) have been recommended to determine the optimal protocol for the retrieval of an epitope (Shi et al., 1996a). Buffer pH

Temperature and duration

120°C for 10 min 100°C for 10 min 90°C for 10 min

pH1–2 Slide # 1 Slide # 2 Slide # 3

pH 6–8 Slide # 4 Slide # 5 Slide # 6

pH 9–11 Slide # 7 Slide # 8 Slide # 9

One reason for interlaboratory differences in the reproducibility of immunostaining results is the use of microwave ovens with significant differences in age, power, construction, and design. Individual laboratories should optimize wattages and duration of heating as well as durations of each of the steps mentioned above; some of them will need to be determined by trial and error. In the absence of standardization, since either false-positive or false-negative immunostaining can occur with any antigen retrieval protocol, the effect of the chosen antigen unmasking method on every individual antigen must be determined using careful

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controls. Appropriate positive and negative controls, as well as the study of fresh-frozen tissue sections, are required to rule out any false-negative or false-positive staining. Duplicate immunostaining will assess reproducibility.

INTRAOBSERVER AND INTEROBSERVER VARIATION IN DIAGNOSIS Pathologists play a key role in the diagnosis of cancer, and their histopathological assessments are accepted as the gold standard. Although not admitted, the process by which a pathologist makes a diagnosis is inherently subjective. A number of factors, including clinical features of the lesion, the clinical impression offered by the surgeon, and the training and experience of the pathologist, play a part in determining the final “signout” diagnosis on which the final treatment decisions depend. These decisions have farreaching consequences in the quality of health care. It is obvious that no other type of error in the medical profession is more important, less understood, and less frequently admitted than fallibility in histopathological diagnosis. Kaugars (1995) has aptly pointed out that the sign-out is written on paper, not on stone tablets. Can the intraobserver and interobserver variations in diagnosis be eliminated? Unfortunately, the answer is no. However, avoidable fallibilities must be avoided. The interobserver variations can be significantly reduced by a joint session behind a microscope in a process of “practical agreement” (Vet et al., 1995). Prior to such sessions, participating pathologists reach consensus on the relevant pathological grading characteristics (theoretical agreement). A theoretical agreement increases the practical agreement between pathologists. Interobserver agreement is definitely improved when pathologists confront each other’s observations and arguments. Even experienced pathologists will benefit by a joint session behind the microscope. Both low-power and high-power microscope observations are useful in markedly minimizing interobserver variation. The characteristics observed at low magnification include atypia, location of immature cells, and stratification/polarization. At high magnification, detailed morphological characteristics, such as location of immature cells and stratification/ polarization (differentiation), nucleus/cytoplasm ratio, hyperchromasia, polymorphous nuclei (cell characteristics), and the location and appearance of mitotic activity, are scored (Vet et al., 1995). Another approach to avoiding interobserver and intraobserver variations and standardizing the diagnosis is the use of computer-assisted analyses. This technology is beginning to be employed in some laboratories (see below).

QUANTITATION OF IMMUNOSTAINING Accurate quantitation of antigens using immunohistochemistry depends upon a linear relationship between the amount of antigen and the intensity of immunoperoxidase-DAB reaction product as well as the percentage of stained cells. Variations in staining intensity will reflect the amount of antigen only if optimal preparatory procedures are used; for example, oversaturation of the chromogen reaction may result in invalid quantitation. Therefore, optimal concentration of DAB should be determined by trials with DAB

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concentrations ranging from 0.1–1.5 mg/ml in Tris buffer containing 0.003% (pH 7.6). Optimal duration of DAB incubation should also be determined by trying incubations for 3–15 min at 37°C. Currently, interpretation of immunohistochemistiy staining in most studies is subjective, qualitative, and nonreproducible. The achievement of quantitative, reproducible results requires standardized immunohistochemical procedures. The development of such a universal procedure is difficult because antigens are not equally affected by a specific processing protocol, including fixation. However, instruments to computerize image analysis have the potential to quantitate immunohistochemically localized antigens. One such instrument is Cell Image Analysis System SAMBA 4000 (Imaging Products International, Chantilly, VA) used in combination with the automatic stainer (OptiMax Staining System, BioGenex Lab., San Ramon, CA) or Tech Mate (Biotek, Santa Barbara, CA). The image analyzer contains software for the densitomeric and RGB (red, green, blue) to HSI (hue, saturation, intensity) colorimetric analysis of cells and tissues (Esteban et al., 1994a). It is based on a light microscope attached to an interactive microcomputer that is capable of high-speed digital image processing for cell measurements. The system includes various software packages for different applications (De Cresce, 1986). It allows immunostained histological sections to be represented as digitized images from which the optical densities of the DAB reaction product over a specific cell part or component (e.g., nucleus) can be quantitated. Bacus et al. (1988) have successfully quantified the estrogen receptor content in human breast tumors. The data showed excellent sensitivity and specificity. Quantitative immunostaining analysis has been performed with a computerized microscopic image processor, SAMBA 200 (SAMBA TITN, Grenoble, France) (Seigneurin et al., 1987). Integrated optical density histograms are provided by the image analysis processor. Mean values and percentage of immunostained cellular surfaces are computerized by the application processor. These computerized systems facilitate multiparametric, accurate, reliable, reproducible, and automatized evaluation of the heterogeneity of the antigenic sites in tumors (Charpin et al., 1989). The advantage of this capability becomes apparent when one considers that tumors showing positive immunostaining are pools of positive and negative cells. To rectify the lack of intra- and interlaboratory reproducibility of immunostaining, Riera et al. (1999) have described the Quicgel method used in conjunction with computerassisted image analysis for quantitation of immunohistochemical data. The Quicgel consists of a cultured cell plate containing a known amount of the antigen, which yields consistent positive staining detected by image analysis. The Quicgel is processed simultaneously with the specimen. This method is based on the assumption that changes in the antigen content of the Quicgel and the specimen vary in parallel during specimen processing. Thus, a decrease in the immunostaining of the specimen during processing is equally demonstrated in the Quicgel. Even without image analysis, Quicgel can serve as a control in immunohistochemical staining. Further application of this protocol for quantitation is awaited. A related approach was used by Ranefall et al. (1998) to quantify images of immunohistochemically stained cell nuclear Ki-67 antigen and cyclin A protein in bladder carcinoma tissue. They combined automatic, computerized image analysis with appropriate controls and reference material. This approach is superior to the automatic method without

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controls. The former method consists of simultaneous processing of embedded cultured controlled cells and tissue sections. Agarose-embedded cultured fibroblasts are fixed, embedded in paraffin, and sectioned at They are immunostained together with paraffin-embedded tissue sections. The image of the control cells serves as a standard control regarding image qualities such as illumination and color properties. Since control cells possess known characteristics regarding antigen expression to be examined in tissue sections, they serve as a means to control and standardize immunohistochemical data (Ranefall et al., 1998). Recently, an automatic color video image analysis system was developed to quantify antigen expression (androgen receptor) (Kim et al.,T999a). This system provides a linear relationship between the antigen content and mean optical density of the immunoperoxidasesubstrate reaction product. Titration of antibody, concentration, and reaction duration of the substrate can be optimized with this system. The imaging hardware consists of a Zeiss microscope, a three-chip charge-coupled-device camera, a camera control board, and a Pentium-based personal computer. It is necessary to properly maintain and calibrate the computerized image analysis system. This is the only way to ensure that the scale of the reported histogram will cover the range of staining intensities obtained in practice (Bacus et al., 1988). In addition, the technician should be knowledgeable about the normal cellular morphology and pathology of the tissue. Counterstaining tissue sections with methyl green in conjunction with the chromogen is also necessary. A relevant question is whether the quantitative measurements obtained with currently available computerized image analysis systems are reliable, accurate, and reproducible and if quantitation of immunostaining reactions offers any real advantage over qualitative evaluations by an experienced pathologist (Rittman, 1998). Many of the image analysis systems are still somewhat rudimentary. Results obtained with various image analyzers are difficult to compare because different hardware and software are used. Unfortunately, in some laboratories quantitation of immunohistochemistry may be used simply to justify the pathologist’s decision in difficult (borderline) cases. The controversy over whether borderline tumors should be classified as benign or malignant and whether they represent a precursor of malignancy remains unresolved. A semiquantitative approach has also been applied for evaluating the concordance between the presence of p53 mutations and immunohistochemical overexpression of this protein in breast carcinomas (Schmitt et al., 1998). The advantage of this approach is that it uses a scoring system based on both the intensity and percentage of stained cells. A limitation of this method is that scoring is performed by examining all low-power optical fields containing tumor, which is time consuming, lacks automation, and is thus subjective, even when scored by more than one observor.

AUTOSTAINERS Presently, immunohistochemistry requires improvements in quality, reproducibility, speed, quantitation, and standardization. Some of these goals can be achieved by using computerized bar code–driven automatic immunostainers that automatically dispense reagents, control washing, mixing, and heating to optimize immunohistochemical reaction

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kinetics and that produce results within 1 hr. Furthermore, the practice of manually staining a large number of slides is tedious and time consuming. Typically, manual hematoxylin-eosin staining (hematoxylin stains nuclei blue and eosin stains cytoplasm pink) is completed in approximately 25 steps. Autostainers save a technologist’s time, which permits him or her to carry out more technically demanding tasks. After the staining protocol has been standardized, the stainer does not require the technologist’s attention during its operation. There are many other advantages to using autostainers. They prevent risk of exposure to certain hazardous reagents (xylene). Some stainers have built-in fume hoods or can be used in a fume hood; in the former case, the need for a large overhead fume hood is eliminated. Another advantage is consistency of the technique that eliminates intra- or interpersonal variations in results. Also, a consistent temperature can be maintained for temperature-sensitive procedures. The space requirement for some stainers (e.g., centrifugal stainer) is smaller than that required for manual staining for some procedures. Efficient stainers use reagents conservatively, thus reducing the amounts needed and the risk of contamination. There are a few limitations to equipping a laboratory with an autostainer. The high cost of most stainers may be prohibitive for a small laboratory. Unavailability of space in such a laboratory is also a possibility. Repairs of this machine are expensive and may take a long time if the service technician lives in another state. Some stainers require the purchase of prepackaged reagents that are also relatively expensive. Moreover, some technologists may object to being restricted to using these reagents. In addition, a staining defect occurring during operation of the stainer is revealed only after staining is complete. On the other hand, if a problem arises during manual staining, the process can be stopped and the problem corrected. But above all, the use of a stainer limits the technologist’s understanding of the actual staining process. There are two types of autostainers: in the first type, the slides are immersed into the reagent; in the second type, the reagent is applied to the slides. Stainers that immerse the slide into the stain (bath stainers) can be either linear or batch design (Earle, 2000). The linear type is based on a carrier mechanism that allows loading of the slides into the slide holders (racks), one at a time, and their sequential immersion into the staining solution. The slide holders are attached to the carrier, which moves at a uniform speed, and the slides exit the stainer one at a time. This type of machine is long, processes ~360–720 slides per hour (12–14 min per slide), and requires water and a drain. It may have a built-in fume hood and slide dryer. Batch stainers move slide holders, each containing several slides, through baths of the staining solution. Programmable batch stainers are now commercially available which use robotic arms to move the slide racks from one position to the next. These stainers can be programmed to agitate the slides in the staining bath. Simultaneous multiple staining can be accomplished in some machines based on this principle. A combined linear-batch stainer is also available, which moves slide containers through a series of stain containers. Each rack may hold a small or large number of slides, and continuous staining is possible. The machine is long, processes 24–66 slides per rack, and the time taken depends on the program. It may be compatible with the coverslipper, and some have built-in fume hoods. The machine may require running water and a drain, and it may have waste collection. There are three types of stainers that apply the reagents to the slides: capillary gap stainers, centrifugal stainers, and flat-method stainers (Earle, 2000).

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Capillary Gap Stainers Capillary gap stainers are based on the principle that the staining solution is forced between the slide and the area around it. Essentially, rotating gears move slides (face downward) along a plane surface that has holes through which the stain is pumped at appropriate intervals. The advancing slides press a switch as they pass each staining station, thus activating a pump. The stain is discarded after the slide moves to the next station, avoiding the contamination of the bulk containers. The machine pumps the stain from the closed bulk containers to the plane surface via small tubing, minimizing reagent evaporation. The capillary gap system can also be used in stainers that use two slides face-to-face to provide the capillary gap. Robotic arms move holders of paired slides to staining, draining, and rinsing stations. Because this system uses very little staining solution, it is recommended for immunostaining large numbers of slides. The machine is ~3 ft long, may be compatible with a cover-slipper, and some may have a built-in fume hood. This system requires prepackaged reagents and may require a drain.

Centrifugal Stainers Centrifugal stainers spray the staining solution onto the slides as they rotate past the spray nozzles in a spinning chamber. The prepackaged reagents are in closed containers with pump tubing, which prevents evaporation and contamination of the chemicals. The machine is smaller than 2×2 feet, stains 12 slides in 6–8 min, requires prepackaged reagents, and may require a drain. It usually does not require a fume hood.

Flat-Method Stainers Flat-method stainers drop staining solutions onto the slide as it lies flat within the stainer. Some stainers employ robotic arms to apply solutions to the slides. This system is in common use for immunohistochemical staining. The machine is long, stains 20–40 slides, depending on the system, in and slides require predeparaffinization. The protocol may require prepackaged or manufacturer’s reagents and a waste container. It is recommended for immunohistochemistry. For additional details about autostainers, see Earle (2000). The following automatic tissue processors and stainers are commercially available. 1. AP 280 Embedding Station, Carl Zeiss, Inc. One Zeiss Drive, Thornwood, NY 10594 2. ATP1 Tissue Processor, Triangle Biomedical Sciences, Inc. 3014 Croasdaile, Durham, NC 27705–47770 3. Cytologix Stainer, Cytalogix Staining System 99 Erie Street, Cambridge, MA 02139 4. Lab Vision Auto Stainer, DAKO Corporation, 6392 Via Real, Carpinteria, CA 93013

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5. Mark 5 HSS Stainer, Diagnostic Products Corporation 5700 West 96 Street, Los Angeles, CA 90045 6. Medite TST40 Slide Stainer, Mopec 13640 Elmira Street, Detroit, MI 48227 7. Medite TPC15 Tissue Processor, Mopec 13640 Elmira Street, Detroit, MI 48227 8. Optimax Consolidated Staining System, Biogenex Laboratories 4600 Norris Canyon Road, San Ramon, CA 94583 9. Protocal Capillary Action Stainer, Biochemical Sciences, Inc. 200 Commodore Drive, Swedesboro, NJ 08085 10. Tissue-Tek. DRS 2000, Slide Stainer, Sakura Finetek USA, Inc. 1750 West 214 Street, Torrance, CA 90501 11. Ventana ES Automated Immunostainer, Ventana Medical Systems, Inc. 1910 Innovation Park Drive, Tucson, AZ 85737

VOLUME-CORRECTED MITOTIC INDEX The volume-corrected mitotic (M/V) index can be used to test for differences between borderline and malignant tumors. This index expresses mitotic activity as the number of mitotic figures per square millimeter of neoplastic tissue in the microscope field. Usually 10 fields are counted at a magnification of 40, which corresponds to of neoplastic tissue in the section. The M/V index has the advantage of not being influenced by the size variation of the microscope field or cellularity of the neoplasm (Haapasalo et al., 1989). Also, this method is easy, relatively rapid, reproducible, inexpensive, and available to all pathologists (Miliaras, 1999). The morphometric formula of the M/V index renders mitotic counts a more reproducible criterion because it avoids some of the limitations, such as differences in microscope field size, of the conventional mitotic index. The M/V index has been used for mitotic counts in many human neoplasms for both diagnostic and prognostic purposes (Lipponen et al., 1990). Recently, Miliaras (1999) has used this index for determining differences in p53 immunoreactivity and the proliferation rate between borderline and malignant ovarian tumors. The M/V index is evaluated as the number of mitoses per 10 hpf (high power field) and is calculated according to the following formula proposed by Haapasalo et al. (1989).

Where n=number of microscopic fields studied (usually 10) Vv=volume fraction of neoplastic tissue (%) as expressed in the area fraction of neoplastic tissue in the microscope field; this is estimated subjectively in the same field in which the mitotic count is made. MI=number of mitotic figures in a microscopic field from the area of highest neoplastic cellularity (during the measurement, the microscope is focused only once). k=coefficient characterizing the microscope: where r is the radius of the microscope field in millimeters (0.255 mm).

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THE GLEASON GRADING SYSTEM Gleason grading is now the most widely used system for grading prostatic carcinoma. This system is an effective tool for prognostication and as an aid in therapeutic decisions for men with prostate cancer. The system is characterized by two major features: (1) it is based solely on architectural pattern but cytological features are not evaluated (Gleason and Mellinger, 1974) and (2) the overall grade is not based on the highest grade within the tumor. The prognosis of prostate cancer is intermediate between the most predominant and the second most predominant pattern of cancer (Fig. 5.3). Consequently, the grades of the most prevalent and the second most prevalent pattern (~5% of the tumor) are added together to obtain a Gleason score (Allsbrook et al., 1999). If the tumor shows only one pattern, the pattern grade is doubled to obtain the Gleason score; for example, for all pattern 3, the Gleason score is 6 (Fig. 5.4). The Gleason score is directly correlated with mortality rates, is a predictor of time to recurrence after surgery, and of response to therapy. Presently, the Gleason score, along with PSA and tumor stage, forms the database upon which radical therapies are recommended (King, 2000). The Gleason score alone suffers from interpretation bias and its accompanying grade errors. Evidence is available indicating a lack of interobserver reproducibility of this score. As expected, interobserver agreement is significantly better among pathologists who learned Gleason grading at a professional meeting or course than among those who had

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not (Allsbrook et al., 2001a, b). The interpretation bias is significantly minimized through a consensus pathological evaluation, while sampling effects are maximally reduced by using an optimal number of biopsy cores. These two remedies, when applied in combination with the Gleason score, result in maximal grading accuracy. Another approach to

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achieve grading accuracy is through using cytokeratin staining in combination with a consensus of three pathologists (Carlson et al., 1998). Although the use of sextant biopsy is an effective technique for prostate cancer diagnosis (Stamey, 1995), and has now become the most common method, it has limitations. Studies have shown that grading based on sextant biopsies, when compared with matched surgical grades, suffers from a significant rate of undergrading. Therefore, biopsy cores should be outside of the anatomical domain of extant biopsies to reduce missed or delayed diagnosis (King and Long, 2000). Because prostate cancer is often multifocal (heterogeneous population of tumor cells), a certain degree of sampling error is expected. The error may result from sampling an area which is either overrepresented with high-grade tumor or, conversely, overrepresented with low-grade tumor as compared to the actual histological grade of the resected prostate (King and Long, 2000). It is not uncommon for well-differentiated cancers to be undergraded and poorly differentiated cancers to be overgraded. To overcome such sampling errors, a directed biopsy must be performed (assuming that an ultrasound-visible lesion is present) or the number and location of biopsies must be increased. In conclusion, the grading error can be significantly reduced by minimizing sampling effects through increasing the number and location of biopsies. The positive role of consensus pathological evaluation in lessening grading errors is equally important. These two remedies will improve the accuracy of Gleason grading of prostate biopsies. As stated above, most patients with prostate carcinoma are diagnosed by core needle biopsy, and tumors are most commonly graded using the Gleason grading system. The Gleason score assigned by the pathologist to the tumor obtained by needle biopsy can profoundly affect the treatment decisions made by urologists, radiation oncologists, and medical oncologists. Although presently the Gleason grading is in common use, many studies indicate inaccuracies in this grading system, with a strong tendency toward undergrading (Carlson et al., 1998). To improve the accuracy of the Gleason grading system, Kronz et al. (2000) developed a free, web-based tutorial program (www.pathology.jhu.edu/prostate). It consists of 20 pretutorial quiz images of prostate carcinoma specimens that were obtained by needle biopsy for grading, followed by 20 tutorial images with text describing the Gleason grading system. Subsequently, pathologists take a posttutorial quiz, consisting of the same 20 images that were used in the pretutorial quiz. This web site tutorial leads to an improvement, especially in the grading of high-grade tumors (Gleason score, 8-10). The web site also improves the grading of tumors with Gleason scores of 5–6 on needle biopsy. An advantage of the web-based media is that it is permanently available for repeated review as opposed to other learning experiences such as lectures.

UNIVERSAL ANTIGEN RETRIEVAL METHOD? Is it practical to develop a universal epitope retrieval method? The answer is no, as the optimal retrieval of each type of epitope requires very specific processing conditions such as fixation, retrieval fluid, and unmasking treatments, including heating, enzymatic digestion, and ultrasound. In terms of preservation and masking, each type of epitope is affected differently by the fixative used, and by its concentration, pH, and the temperature and duration of fixation. As long as different fixatives are used in different laboratories,

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interlaboratory standardization of immunohistochemistry will remain elusive. Storage of tissue in the fixative, as well as sections of the fixed tissue mounted on glass slides, influences epitope retrieval. Some evidence indicates the potential loss of immunostaining in stored paraffin sections, especially with prolonged durations at room temperature (see page 84). It is not uncommon for pathology laboratories of small hospitals to mail unstained slides to other laboratories for immunostaining and second opinions. Epitope retrieval depends upon epitope preservation and hence on the effects of fixation or chemical fixation. In some cases, the fixation history of archival tissues is not known, making the rational choice of epitope retrieval methods problematic. Another problem is that similar antibodies obtained from different sources may vary in their sensitivity and specificity. The procedure used to dry sections on a glass slide may also affect the degree of immunoreactivity. This is exemplified by nuclear antigens (e.g., proliferating cell nuclear antigen) that show decreased immunoreactivity when sections are hot-plated onto glass slides (Hall et al., 1990). The composition, pH, and amount of retrieval solution influence the degree of epitope retrieval. In addition, the type of heating used (microwave, autoclave, steamer, hotplate, and conventional oven) affects the degree of epitope retrieval. Overwhelming evidence indicates that all types of epitopes are not equally unmasked with any one source of heating. In other words, certain types of epitopes are maximally retrieved with microwave heating, while some other types are best retrieved with an autoclave or a steamer. In addition, ultrasound treatment may be efficient for unmasking certain epitopes. Furthermore, optimal retrieval of certain epitopes is obtained with combined treatments such as microwave heating-ultrasound or enzymatic digestionmicrowave heating. Caution is warranted in the use of enzymatic digestions, as this treatment can adversely affect cell morphology and antigenicity. The temperature used during epitope retrieval is important, and if microwave heating is used, so is the amount of water load and its exact location in the oven. The extent of epitope retrieval is also section-thickness dependent. It should be noted that the same processing conditions show different retrieval efficiencies of similar epitopes in animals of different species and ages. Whether a protein molecule is glycosylated or not affects its unmasking with an epitope retrieval protocol. For example, it has been demonstrated that the more glycosylated human placenta fibronectin has a higher resistance to protease treatment, and thus a reduced epitope retrieval, than the less glycosylated plasma fibronectin (Zhu et al., 1984). Even different isomers of an antigen may require different epitope retrieval methods. In addition, the degree of epitope retrieval is ethnicity-dependent. Also, optical microscopy, fluorescence microscopy, and electron microscopy require different methods of fixation and epitope retrieval. A pretreatment that facilitates the recognition of a given epitope may destroy other epitopes in the same antigen or in other antigens. In conclusion, although a universal epitope retrieval method is almost impossible to formulate, the development of a general strategy for adequate retrieval of epitopes is feasible. Such an approach is presented in Chapter 8.

CALIBRATION OF MICROWAVE OVEN As stated earlier, lack of a standardized antigen retrieval method results in intra- and interlaboratory variability in immunostaining results. The following method of microwave

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oven calibration is a step toward obtaining reliable immunostaining (Tacha and Chen, 1994). This approach avoids repeated interruption of oven heating to replenish the antigen retrieval fluid. The method essentially establishes the time to boiling point, after which the setting is adjusted to maintain a simmering temperature. At such mild temperatures, the separation of sections from the slide is less likely. Place the jar containing 250 ml of antigen retrieval fluid and slides in the center of the microwave oven, and set the oven on high power (800 W) for 2–3 min, until the fluid begins to boil. Turn off the oven and record the exact time it took to achieve boil. Set the oven on low power (~300 W) for 7–10 min, and adjust the setting so that the oven cycles on and off every 20–30 sec and the fluid boils for ~5–10 sec/cycle. Also, note this setting. The following formula can be used to determine the power setting: S = 250/P × 10, where S is the oven power setting and P is the output power of the oven. For example, if the oven output power is 800 W, the power setting for antigen retrieval (S) will be S= 250/800 × 10= 3.1. Therefore, set the oven on 3 and heat at 100°C for 7–10 min to achieve antigen retrieval, depending on the antibody used. Microwave ovens with temperature readouts are commercially available (Energy Beam Science, Agawam, MA). Their power output is regulated by a temperature feedback mechanism and timer, so that both temperature and time can be monitored. They can also be used for fixation and accelerated immunostaining.

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Chapter 6

Antigen Retrieval

POSSIBLE MECHANISMS OF ANTIGEN RETRIEVAL Heating, especially microwave heating, effectively unmasks a wide variety of epitopes (e.g., Fig. 6.1). However, in some cases epitopes are best unmasked by methods other than heating. Such alternate procedures include enzyme digestion and treatment with detergents. Furthermore, the retrieval of some epitopes is accomplished with a combination of methods such as microwave heating–enzyme digestion, microwave heating–sonication, or microwave heating–EDTA. In fact, variations of the heating method may affect epitope retrieval differentially. These variations include microwave heating, autoclaving, pressure cooker, water bath, and even a hot plate. Comparative studies using different heating methods demonstrate that an antigen under study is retrieved optimally by only one of the heating variations, and that heating method may be other than microwave heating (see pages 153–154). However, it is most likely that each heating method is equally effective in antigen retrieval provided it is used under its optimal conditions. Nevertheless, microwave heating is presently the most commonly used procedure to retrieve antigens in formalin-fixed tissues. These observations suggest that multiple mechanisms are responsible for epitope retrieval. A number of possible mechanisms responsible for epitope unmasking have been advocated, including breakage of protein crosslinks introduced by formaldehyde, denaturation of proteins to reveal previously masked epitopes, and unmasking of epitopes by removing calcium ions. Major mechanisms are discussed below. Fixation with formaldehyde tends to alter the conformation of the protein molecule, making it unrecognizable by the antibody. Antigen retrieval treatments may restore the original, native protein structure, reestablishing the three-dimensional structure of the protein, or coming very close to that state (Shi et al., 2001). In other words, antigen retrieval treatments may renature the protein structure that was altered during fixation. However, direct evidence supporting the revival of the native conformation of the epitope with antigen retrieval pretreatment is lacking. On the other hand, available evidence supports the occurrence of the breakage of protein crosslinks, which allows the antibody access to the antigen. Conventional heat, dry or steam, breaks down reversible protein-protein, protein–nucleic acid, and protein-carbohydrate crosslinks introduced by formaldehyde and thereby unmasks the epitopes, as well as allowing the antibodies access to the epitopes. It is well known that most, if not all, crosslinks formed during formaldehyde fixation are destroyed upon heating even at 37°C 117

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for a prolonged time (e.g., 48 hr) or at higher temperatures (e.g., 80–100°C) for very short durations, that is, in minutes. Specific peptide-bond cleavage by microwave heating in weak acid solutions is a well-established method in protein chemistry (Wu et al., 1992). The specific cleavage sites of peptide bonds are located at the carboxyl- and amino-terminal ends of aspartyl residues along the peptide chain. Thus, heat can free epitopes from other proteins and attached molecules. That heat is not the only method to unmask epitopes is exemplified by enzyme digestion or detergent treatment. The exact mechanism responsible for epitope retrieval with ultrasound is not clear, although intense heat is produced for an exceedingly short duration. It is known, however, that ultrasound and/or heat decreases the amount of negative charges on the cell surface (Joshi et al., 1983; Adler et al., 1988). Mechanical vibrations of molecules caused by ultrasound and heat are thought to unfold the protein molecule and to expose the epitopes. The mechanism underlying unmasking of epitopes with digestive enzymes is better understood. Enzymes such as trypsin II, used in epitope retrieval, are powerful, tested protein-digestive molecules. They are known to digest proteins and break down protein crosslinkages introduced during formaldehyde fixation. As a result, the tight network surrounding the epitopes is dismantled, allowing access of antibodies to the epitopes. If antigen retrieval with protease digestion must be carried out, 100 mg of trypsin in 100 ml of Tris-buffered saline (pH 7.8) can be used for 15–20 min at 37°C.

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Some information is available on the possible mechanism underlying masking of epitopes in the presence of calcium ions. Such a masking of epitopes and their unmasking are discussed in detail on page 120.

Nonthermal Effects of Microwave Heating Like conventional heat, microwave heat breaks down protein crosslinks. The most common explanation presented in the literature for unmasking epitopes with heat in the microwave oven is hydrolysis of protein crosslinkages. However, the effects of microwave heating on the tissue sections resulting in epitope retrievals are exceedingly complex. Heat alone may not be enough to explain the effects of microwaves on epitope retrieval. This view is supported by the observation that microorganisms are killed at lower microwave temperatures or with shorter exposures than those required when conventional heat is used (Chipley, 1980). Furthermore, because microwave heating also enhances immunostaining of ethanol-fixed tissues, it is apparent that such heating unmasks epitopes by a mechanism other than or in addition to breakage of protein crosslinks because this coagulative fixative does not introduce crosslinks. The above-mentioned evidence indicates that in addition to the direct thermal effect of microwaving, microwave heating facilitates epitope retrieval by another simultaneous mechanism. The microwave energy irradiated on the tissue sections in various liquid media is lost or absorbed by the samples by two mechanisms: ionic conduction and dipole rotation. Both effects occur simultaneously to account for the phenomenon of rapid heating (Kingston and Jessie, 1988). It is thought that microwaves unfold protein molecules, exposing the epitopes by subjecting the molecules, at least polar molecules such as water and polar side chains of proteins, to rotational movement. As a result, these molecules reach to a high energy level, unmasking the epitopes. It is known that microwaves interact with dipolar molecules by (1) imparting kinetic energy and raising temperature and (2) altering electric fields. Microwaves induce dielectric fields, causing dipolar molecules to rapidly oscillate 180 degrees. In other words, these molecules oscillate at the frequency of 2,450 MHz or at about 2.5 billion cycles per second. Thus, microwave action is also due to rapid oscillation along the axes of asymmetrical molecules such as water, proteins, and fatty acids, which behave as dipoles in an attempt to reorient their positive and negative poles to keep up with the rapidly changing electrical fields generated by microwaves (Salvatorelli et al., 1996). It has been shown that the oscillating electric field causes cell poration (Chang, 1989). It is also known that microwaves irreversibly alter the plasma membrane, with subsequent changes in ion transport, breakdown of hydrogen bridges and secondary bridges, alterations in protein hydration, and release of bound water. All of these phenomena explain why microwaves exert a different effect than that of conventional heat. It is apparent that not only the thermal but also the nonthermal component of microwaves deserve consideration as effective epitope retrieval factors. Evidence indicates that microwaves affect the kinetics of conformation changes of proteins such as (Bohr and Bohr, 2000). It is thought that even approximately a few GHz can excite protein molecules. Consequently, the kinetics of conformational changes of the protein molecule are enhanced, and this denaturing effect is

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nonthermal. In fact, microwave irradiation can cause folding or unfolding of protein molecules. However, additional evidence is needed to substantiate the role of the nonthermal effect of microwave irradiation in antigen retrieval.

EFFECT OF ENDOGENOUS CALCIUM ON ANTIGEN MASKING An understanding of the interaction between the antigen (epitope) and the antibody visualized immunohistochemically can be attempted by considering at least the role of formalin fixation, antigen retrieval fluid, and heat treatment. How each of these three factors affects the molecular structure of the antigen or the epitope is the fundamental question. The significance and relevancy of this question are apparent because an antibody recognizes the corresponding epitope based on the molecular structure of the latter. The following discussion considers the possible role of calcium in masking antigens during fixation with formaldehyde; it is based primarily on studies carried out by Morgan et al. (1994, 1997a, b), Shi et al. (1997, 1999a), and Taylor et al. (1996a, b). The role of other factors in antigen masking and retrieval is discussed elsewhere in this volume. Endogenous calcium is an important factor in epitope masking. Biochemical studies indicate that calcium binding induces a conformational modification of the protein molecule, resulting in either a reduced antigen-antibody recognition effect (e.g., for thrombospondin) (Wilson, 1991) or the reverse effect (for protein C, a vitamin K–dependent enzyme involved in blood coagulation) (Wakabayashi et al., 1986). It has been proposed that removal of calcium by chelation significantly modifies the thrombospondin conformation (Dixit et al., 1986). These changes may expose epitopes necessary for the binding of certain monoclonal antibodies. This and other evidence indicates that calcium-induced changes in the conformation of different proteins may result in negative or positive detection of immunogenicity. The ability of some monoclonal antibodies, but not all, to recognize their corresponding epitopes is calcium-dependent under certain conditions. Different antibodies respond differently to the calcium-induced modification of the same protein. In relation to fixation with an aldehyde, possible mechanisms responsible for masking or unmasking epitopes as a result of tissue-bound calcium and calcium chelation, respectively, are detailed below. One of the major effects of formaldehyde fixation is the generation of a large number of hydroxymethyl groups through selective interactions with various functional groups (e.g., active hydrogens on aromatic rings, primary and secondary amines, and hydroxyl and sulfhydryl groups) in proteins. The hydroxyl component of the hydroxymethyl groups is thought to be reactive, depending on which of these functional groups the formaldehyde is bound to. Such an active hydroxyl component could form a coordinated bond with calcium ions (Fig. 6.2). Thus, proteins fixed with the aldehyde may become complexed with calcium ions that are abundant in animal tissues, reaching levels on the order of 2 mM in the cytoplasm of eukaryotic cells. These complexes mask epitopes to a variable degree. Calcium complex formation with proteins in this state is likely to be quite strong, involving four to eight coordinate bonds. Therefore, a considerable amount of energy (heat) is required to release the calcium ions from this cagelike complex. Based on this observation, calcium released from this complex requires hightemperature heating in combination with a calcium chelating and/or precipitating agent such as EDTA, EGTA, citrate buffer, or urea. Because these reagents are chelators of divalent

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metal ions, epitope unmasking can be achieved by exposing the sections to such treatments. Figure 6.3 shows the disruption of some coordinate bonds at a high temperature in the presence of EDTA, which results in antigen retrieval. Also, an inorganic salt such as sodium carbonate is expected to remove calcium by preferential precipitation. It is interesting to note that microwave heating also causes changes in metal ion transport through the plasma membrane.

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Further evidence supporting the role of calcium in antigen masking was provided by Shi et al. (1999a). They exposed frozen tissue sections to 50 mM (pH 7.1) overnight at 4°C and demonstrated a significant loss of immunostaining or altered staining pattern of antigens such as thrombospondin and Ki-67 compared to controls not exposed to Such a loss of staining can be partially recovered by incubating the sections in EDTA, substantiating the role of calcium in antigen masking. The antigen masking effect of calcium has also been demonstrated by adding to antigen retrieval fluid such as EDTA (Kim et al., 1999b). The disodium salt of EDTA (in common use) binds one divalent metal ion only, and the addition of molar excess of calcium ions would eliminate the antigen retrieval effect of EDTA. The role of pH in calcium-related effects on antigen unmasking is controversial. According to Morgan et al. (1997b), two different mechanisms are involved in antigen retrieval at acidic and alkaline pH levels. Under acidic conditions (pH 1–3), instead of chelation, high concentrations of hydrogen ions dissociate calcium complexes and/or breakdown protein crosslinkages introduced by formaldehyde. On the other hand, in an alkaline environment (pH 8.0), chelation of calcium is responsible for antigen retrieval and can be carried out with a chelator. Dixit et al. (1986) had also proposed earlier that the removal of calcium by chelation modifies the conformation of protein molecule as an unrolling or unraveling of the large domains, resulting in the exposition of epitopes necessary for the binding of certain monoclonal antibodies. A somewhat different interpretation of the relationship of aldehyde fixation with the masking of antigens with calcium-protein complexes is reported by Shi et al. (1999a). According to this point of view, although calcium-induced modification of the protein molecule does occur and can be demonstrated immunohistochemically, it is independent of formalin-induced crosslinking. Addition of calcium chloride can reduce or alter immunostaining, but it is not related to the pH of this solution. In conclusion, the effects of calcium bound to tissue are highly complex, for calciuminduced molecular modification may diminish antibody-antigen recognition or enhance this effect. It is known that the ability of some monoclonal antibodies to recognize their corresponding epitopes is calcium-dependent under certain conditions. The presence of citrate buffer is not necessary to restore antigenicity, provided an appropriate pH is present. The effect of bound calcium is not the only factor responsible for antigen masking. In addition, modification of a protein molecule also occurs due to crosslinking introduced by formalin fixation, causing antigen masking. Calcium binding to protein molecules influences the immunoreactivity of some epitopes, while others are not affected. On the other hand, heat-induced hydrolysis of protein crosslinks is the primary mechanism responsible for epitope unmasking. Possible mechanisms responsible for epitope retrieval by heat treatment are summarized below. In summary, reasoned arguments have been presented in support of several mechanisms responsible alone or in combination for antigen retrieval by heating; denaturation and hydrolysis, self-assembly of unfolded protein chains and the subsequent restoration of antigenic sites, chelation of calcium complexes, and unfolding of protein structure by metallic salts or urea solutions through dissociation of hydrogen bonds or through the loss of diffusable blocking proteins (Macintyre, 2001). In this respect, the role of residual paraffin in the sections is unclear.

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USE OF ETHYLENEDIAMINETETRAACETIC ACID (EDTA) FOR ANTIGEN RETRIEVAL Epitope unmasking can be achieved by exposing sections to high temperature in combination with a calcium-chelating or precipitating agent such as EDTA or EGTA. In fact, maximum antigen retrieval induced by heat is obtained in the presence of EDTA. Such a treatment leads to the extraction of calcium ions tightly complexed to formaldehyde-fixed tissue sections. EDTA solution compared with other antigen retrieval fluids, including sodium citrate buffer, is more effective in certain cases in augmenting not only staining intensity but also the number of positively stained cells. It has been reported that EDTA solution, when combined with heating in a pressure cooker, is more effective than citrate buffer or Tris-HCl buffer in retrieving Ki-67 antigens in gastric and breast cancer tissues (Kim et al., 1999). The intensity of immunostaining of the sections treated with the EDTAheat combination depends on the pH of EDTA. Strong staining is achieved at pH 3 and at neutral to high pH levels, but the staining intensity decreases at pH 4 and 5. Another recent study also supports the mediation role of EDTA in unmasking antigens (Röcken and Roessner, 1999). In this study thin sections of aldehyde-fixed, Eponembedded human biopsy tissues were treated with 1 mM EDTA, using a heated water bath. This combined treatment resulted in excellent immunogold staining of amyloid (Fig. 6.4).

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Because heating in water alone did not improve immunostaining, it is concluded that epitope retrieval is mediated not only by heat and rehydration but also by the presence of a chelating agent such as EDTA. However, caution is warranted in using EDTA, which may adversely affect cell morphology because it is a strong oxidant.

ANTIGEN RETRIEVAL WITH HEAT TREATMENT Theoretically, any method of heating should unmask epitopes. Although most antigens can be detected after heat treatment, some may be destroyed, and others may remain masked. The temperature, changes in temperature during heating, and the duration of heating critically influence antigen retrieval. Different tissues, fixatives, and durations of fixation require specific temperatures and durations of heating. Therefore, pilot studies should be carried out to determine optimal heating conditions. Similar staining intensities are achieved by the following heating conditions irrespective of the heating method used: 100°C for l0min, 90°C for 30min, 80°C for 50 min, and 70°C for 10hr (Shi et al., 1995a). Heating at 100°C for l0min (two cycles of 5min each) is recommended for retrieving most antigens, except those damaged by high temperature. In the latter case, lower temperatures for extended durations can be used. Overfixed tissues require high temperatures and/or extended durations of heating. In some cases, durations of heating longer than 10 min on a full-power setting may cause background staining. Repeating the boiling cycles is more effective than extending the boiling duration. This can be accomplished by removing the slide jar from the microwave oven after each run and placing the slides in a new jar containing the fresh retrieval fluid at room temperature, followed by again placing the jar in the oven. Compared with high-power microwave outputs, medium wattage (e.g., 450 W) may yield better sensitivity, probably due to optimal thermal effects and hence optimal oscillation of dipolar molecules.

ADVANTAGES OF HEATING The recognition of many types of antigens by antibodies is facilitated using hightemperatures. Theoretically, high-temperature heating disrupts protein crosslinks introduced by formaldehyde, causes peptide cleavage, and alters protein tertiary structure, resulting in the exposure of masked epitopes for immunostaining. The retrieval of some types of antigens can be accomplished only by heating. Heating allows the use of antibodies that heretofore could not be employed on sections of tissues fixed with formaldehyde and embedded in paraffin. The heat-induced antigen retrieval procedure lowers the detection threshold for the antigen and improves signal-to-noise ratios; this is true for both monoclonal and polyclonal antibodies. In addition, the development of heating methods permits abandoning the use of frozen tissues, which are difficult to process and study. Another advantage of heating is that it allows the detection of antigens resistant to proteolytic enzyme digestion and retrieves antigens on sections of tissues left in formalin for prolonged durations. Generally, heat treatment is superior to enzyme digestion for antigen retrieval. For example, when using polyclonal anti-kappa and anti-lambda antibodies (1:500),

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immunoglobulin light-chain immunostaining was better after microwave heating than after trypsin digestion (Ashton-Key et al., 1996). Deparaffmized sections were heated on full power in a 750 W microwave oven for 22min, while others were treated with 0.1% trypsin for 10 min at 37°C. Similarly, in human biopsy tonsil tissue stains better after microwave heating than after trypsin treatment (Fig. 6.5). If the retrieval of an antigen type is adversely affected by heating, protease digestion of sections is the optimal pretreatment. However, the preservation of cell morphology is generally better in heat-treated than in enzyme-treated sections, especially when extended durations of digestion are employed.

HEATING METHODS Different heating systems, such as microwave ovens (Shi et al., 1991), pressure cookers (Norton et al., 1994; Miller and Estran, 1995), microwave heating–pressure cookers (Taylor et al., 1995), autoclaves (Bankfalvi et al., 1994), steamers (Taylor et al., 1995), water baths (Kawai et al., 1994), and electric hot plates (von Wasielewski et al., 1994), in combination with antigen epitope retrieval fluids, have been used with various degrees of success. Although each of the methods has minor advantages and limitations, they yield a fairly similar degree of antigen retrieval when appropriate heating conditions are provided. All the processing conditions must be adjusted for a specific study. Such conditions may differ from those most widely cited in the literature or recommended by the manufacturers. The choice of the heating method also depends on equipment availability. A recent comparative study of the following five heating methods using 21 antibodies also demonstrated that they produce similar intensities of immunostaining of retrieved antigens provided the heating durations are adjusted appropriately (Taylor et al., 1996b). However, heating methods Nos. 2, 3, and 4 (given below) yield better results. Advantages and minor limitations of the heating methods are listed below. 1. Microwave heating for 10 min, carried out in a standard, simple, inexpensive, and widely available microwave oven, is the fastest procedure. Total time required (including set up of preheating, actual retrieval process, and cool down) is 25 min. A limitation is possible boiling over, resulting in the loss of antigen retrieval fluid. Consequently the level of the fluid must be checked every 5 min. If necessary, more fluid can be added after the first 5 min to avoid drying the tissue sections. If more fluid is needed, this is the result of boiling over, not evaporation. To catch any boiled-over fluid, the slide jar should be placed within a larger jar which contains deionized water. In addition, the presence of hot or cold spots in the microwave oven is not uncommon when several isolated jars containing the slides are placed at random in the oven, a practice that leads to reduced reproducibility. This method is also difficult to standardize. The microwave oven (900 W, 2,450 MHz) is set at maximum power for two cycles of 5 min each.

Step-by-Step Protocol Determine the optimal pH of antigen retrieval solution for each antigen. Citrate buffer (0.01 M) adjusted to pH 6.0 with HC1 is used widely. Determine the desired temperature based on the type of tissue and antigen under study. For fatty tissues, 90°C is recommended; adjust the duration of heating accordingly. Place slides in plastic Coplin jars containing the

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antigen retrieval solution in the center of the rotary plate in the microwave oven to ensure uniform heating of slides. Cover the jars with loose-fitting caps. Turn on the microwave oven and check the temperature of the retrieval solution with a temperature probe. Use a maximum power setting of 7–10. Start timing the antigen retrieval duration when the retrieval solution begins to boil. As an average, a total retrieval duration of 10 min, divided into two 5-min cycles with an interval of 1 min between cycles to check the solution level in the jars, is recommended. If needed, fresh retrieval solution from an adjacent jar can be added to the jars containing the slides. Alternatively, distilled water can be used to replenish the retrieval solution. The objective is to keep the slides fully immersed in the solution before restarting the oven. Alternatively, the duration can be based on previously determined time for sections known to contain the antigen under study. Remove jars from the oven, and cool the sections for 20 min at room temperature. The slides are ready for immunostaining after being rinsed in 0.5 M PBS (pH 7.4) for 5 min. Do not reuse the antigen retrieval solution. Some of the above-mentioned steps are automatically controlled by the H2550 Laboratory Microwave Processor. 2. Microwave heating for 20 min is the same as Method 1 except that the heating is employed four times for 5 min each. Total time required is 35 min. Improved immunostaining of many types of antigens can be achieved by extending the heating time. The procedure requires attention for 20 min to check the fluid level, and occurrence of hot or cold spots may complicate the procedure. 3. Pressure cooking. Although microwave heating is widely used for antigen retrieval, this system does not raise the temperature of an aqueous buffer above 100°C, even though this temperature is reached rapidly. In contrast, an advantage of the pressure cooker is that, if required, temperatures of 115°C or higher (superheating) can be achieved. Other advantages of heating in a pressure cooker include short duration of heating, better reproducibility of results with large batches of slides, the ability to use metal slide racks, and economy of time and equipment cost (Norton et al., 1994).

Step-by-Step Protocol Fill to approximately one-third capacity of a domestic pressure cooker (103kPa/15 psi) with 0.1 mM citrate buffer (pH 6.0). Bring the buffer to a boil using an electric hot plate, without sealing the lid. Quickly place metal racks containing rehydrated sectionmounted glass slides into boiling retrieval buffer, and seal the pressure cooker. Bring the cooker to full pressure. Start timing when the pressure indicator valve reaches the maximum (~4 min). The optimal duration of pressurized boiling is 1–2 min. Depressurize the cooker and cool it under running tap water. Remove the lid, and add cold tap water to replace the hot retrieval buffer. A duration of 15–20 min is required to cool the cooker. Wash the slides in several changes of 0.05 M PBS (pH 7.4) prior to immunostaining. At no time during this processing are the slides allowed to dry out. The pressurized boiling (120–122°C) longer than ~2min will progressively degrade the cell morphology. 4. Pressure Cooker–Microwave heating The pressure cooker–microwave heating method is simpler than the autoclave procedure and more efficient than microwave heating alone. The pressure cooker does not require checking the level of the antigen retrieval solution during heating in the microwave oven, and a large number of slides can be loaded simultaneously. In addition, the pressure

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cooker does not develop cold spots because it is a larger container. The limitation is that a slightly longer duration (~45 min) is required than that for microwave heating alone.

Step-by-Step Protocol Place slides in three plastic staining jars, each containing 24 slides and antigen retrieval solution, and transfer them into a plastic pressure cooker (Nordieware, Minneapolis, MN) filled with 600 ml of distilled water; this amount of water is one-half the capacity of the cooker. Make sure that the jars stand stably in the water (Taylor et al., 1996b). Transfer the pressure cooker into a microwave oven (model R-4A46) which is equipped to switch one power level setting to another automatically. Place the cooker in the center of the microwave oven. Set the oven at maximum power (900 W, 2,450 MHz) for 15 min to boil the water, then switched to a 40% power setting for an additional 15 min to maintain mild boiling (simmering). Remove the cooker from the oven, and allow it to cool for 15 min. 5. Autoclave heating, like pressure cooking, provides superheating at temperatures higher than 100°C. Hydrated autoclaving eliminates the need to adjust the volume of the antigen retrieval solution. Other advantages include the use of larger volumes of the retrieval solution, which gives a uniform heating pattern and allows the heating of a large number of slides in a single batch. In this method high-intensity immunostaining is achieved, and the cold spots are absent. Hydrated autoclaving of slides, even in deionized water, is thought to be more effective than either microwave or water bath heating (Shin et al., 1991). It is known that heat denaturation of antigens is effective when the protein is hydrated, whereas dehydrated protein is extremely resistant to heat denaturation. Minor limitations are that the autoclave is expensive and may not be available in some small laboratories. Also, the total time required to complete heating is ~45 min. Care should be taken in handling owing to the pressure in the autoclave. The results of autoclave antigen retrieval are shown in Figure 6.6 (Plate 3C, D, E).

Step-by-Step Protocol Place slides in Coplin jars containing antigen retrieval solution that has been previously heated at 80°C. Set the jars in the center of a stainless steel autoclave equipped with a 1,850 W heating filament. Tightly close the door of the autoclave as required by the instructions, and heat at 120°C for 10 min at 15 psi. Cool down with running tap water for 20–30 min, then rinse the sections with 0.05 M PBS at room temperature and immunostain. 6. Steam heating for 20 min over boiling water. Total time required is 35 min. This method has the advantage that loading and unloading of slides into various carriers for automated use is not required. It needs relatively small amounts of the antigen retrieval fluid, does not have cold spots, and is inexpensive. This approach is well suited to process a large number of slides simultaneously and thus saves considerable amount of time. Although originally designed for autostaining, it can be used manually. A minor limitation is that preheated steam is needed. Slides are set into the TechMate slide holder (Biotek, Santa Barbara, CA), with antigen retrieval fluid in the capillary gap, and are heated by steaming over boiling water. In contrast to microwave heating, steam treatment heats slides slowly to a uniform temperature. This avoids boiling the antigen retrieval fluid and minimizes section detachment from slides. Steam heat used in combination with EDTA and protease digestion has been

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reported to be superior to other antigen retrieval techniques for the immunostaining of cytokeratin (using antikeratin antibody) in select cases of prostatic carcinoma (Iczkowski et al., 1999). This approach minimizes the diagnostic ambiguity often encountered when using this antibody. Figure 8.8 shows clearly the superiority of the steamEDTA-protease method over protease treatment alone in detecting acinus with high-grade prostatic intraepithelial neoplasia. However, for a similar study, the hot plate method is preferred (see below). 7. Hot plate heating is the simplest and fastest heating method to retrieve antigens in sections of formalin-fixed and paraffin-embedded tissues. Certain antigens are optimally retrieved with the hot plate heating procedure than with enzyme digestion or microwave heating methods. The hot plate heating method is also most effective in retrieving certain antigens in tissues fixed with formalin for as long as 1 month. The retrieval of basal cell–specific, anti-high-molecular-weight cytokeratin (HMCK) in sections of radical prostatectomy specimens fixed in formalin and embedded in paraffin has been accomplished by using the hot plate method; monoclonal antibody clone raised against human stratum corneum was used in this study (Varma et al., 1999). Step-by-Step Protocol

Sections mounted on a glass slide are placed in a beaker containing 1,000 ml of 0.2 M citrate buffer (pH 6.0) and heated on a hot plate (Corning, Utica, NY) for l0min at 100°C, and then allowed to cool at room temperature for 20min. 8. Equally good results, if not better in some cases, can be obtained with overnight treatment of tissue sections in Tris buffer (pH 9.0) in a conventional oven at 70–80°C

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(Koopal et al., 1998). This method has been successfully used for retrieving antigens such as estrogen and bcl-2 in cervix tissue and lymph node, respectively. Hot oven heating can also be tried in a humidified chamber. 9. Another antigen retrieval method is hot water bath heating at 90°C for 120min. Total time required is about It is simple and inexpensive, processes a large number of slides each time, and does not require replenishment of the antigen retrieval fluid. The method has been successfully used for the immunostaining of p53 and proliferating cell nuclear antigens (PCNA) (Kawai et al., 1994). For p53 and PCNA retrieval, 0.01 M PBS (pH 7.2) and 0.01 M citrate buffer (pH 6.0), respectively, are recommended (Fig. 6.7). The use of this method is limited since it takes much longer time to complete. Hot oven heating in a humidified chamber can also be tried.

Mechanism of Epitope Retrieval by Microwave Heating The effects of microwave heating on the tissue sections that result in epitope retrieval are exceedingly complex. A full understanding of the actions of microwaves at the molecular level to facilitate epitope retrieval is lacking. At least two mechanisms need to be considered: heat and kinetic energy of the oscillating electromagnetic field. Both possibilities are discussed below. The most commonly accepted point of view is that heat is responsible for unmasking the epitopes. In fact, Battifora (1996) has introduced the phrase heat-induced epitope retrieval (HIER). Heating at 100°C is a powerful treatment that can unmask hidden, buried, or crosslinked epitopes. Heat can be provided not only by a microwave oven, but also by an autoclave, a pressure cooker, steam, or a hot plate. A consensus on which method of heating is most effective in the retrieval of all types of epitopes is lacking. Therefore, some

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factor or factors in addition to heat also become relevant. It is also known that treatments other than heat can also unmask epitopes. Such treatments include enzyme digestion and exposure to detergents. The aforementioned observations indicate that heat is not the only mechanism responsible for epitope retrieval. Therefore, the question arises, is the heat in the microwave oven the only factor or the primary factor that facilitates epitope retrieval? Is it possible that in addition to heat, kinetic energy plays a part in epitope unmasking (personal communication, A. S.-Y. Leong)? It is known that the microwave electromagnetic field causes polar molecules in the tissue to oscillate at a rate of 2.45 billion cycles per second, enough to disrupt protein crosslinking and unmask hidden epitopes. In addition, the fact that ultrasound treatment, which generates heat for an exceedingly short duration, also unmasks epitopes, suggests that factors other than heat may also be important in explaining the phenomenon of epitope retrieval in a microwave oven. The following explanation may further understanding of the release of thermal energy and heat in a microwave oven. Microwave energy is a nonionizing radiation (frequency, 300–300,000 MHz) that causes molecular motion by migration of ions and rotation of dipoles. Dipole rotation refers to the alignment, due to the electric field, of molecules that have either permanent or induced dipole moments in both the solvent and specimens. As the field intensity decreases, thermal disorder is restored, which results in thermal energy being released. At 2,450 MHz (the frequency used in commercial systems), the alignment of the molecules followed by return to disorder occurs times per second, resulting in rapid heating. However, the absorption of microwave energy and its release as heat are strongly dependent on the relative dielectric constant (relative permittivity) and the dipolar status of the medium. The relative permittivity is the following ratio: material dielectric constant: vaccum dielectric constant. The greater the relative dielectric constant, the more thermal energy released, and the more rapid the heating for a given frequency (Camel, 2001). Due to the particular effects of the microwaves on matter (namely dipole rotation and ionic conductance), heating of the section, including its core, occurs instantaneously, resulting in rapid breakdown of protein crosslinkages. Furthermore, the extraction and recovery of a solute from a solid matrix with microwave heating is routinely obtained in the field of analytical chemistry (Camel, 2001). However, a definite, full explanation of the effects of microwave heating on the molecular aspect of antigen retrieval is awaited.

Duration of Microwave Heating The duration of microwave heating to retrieve epitopes depends on the type of concentration of the aldehyde used for fixation, duration of fixation, and the temperature in the microwave oven. The higher the concentration of the fixative and the longer the duration of fixation, the higher the temperature and the longer the duration of microwave heating required for epitope retrieval. The oven temperature is controlled using the temperature probe of the oven and is checked with a thermometer. In a microwave oven with 720 W power, the boiling point for the epitope retrieval fluid in the Coplin jar is reached in 140–145 sec (Shi et al., 1994). The time it takes to reach a temperature of 55°C is ~76sec. At 720 W, 5–10 min heating time is recommended, which can be divided into two 5-min cycles with an interval of 1 min between cycles to check on the fluid level in

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plastic or glass jars. If necessary, more fluid at the same pH can be added after the first 5 min to avoid drying the tissue sections. The jars can be covered with perforated cling film to minimize evaporation. Alternatively, 1 hr at 55°C or 30–120 min at 90°C (not boiling) can be used but is not preferred. With long durations (24 hr–3 years) of fixation with formaldehyde, 20 min exposure to heating at 100°C is recommended (Fig. 6.8) (von Wasielewski et al., 1994). A thorough washing of slides after microwave heating in the presence of epitope retrieval fluid and before incubation is essential to avoid background staining.

Antigen Retrieval in a High-Pressure Microwave Oven Although microwave heating, pressure cooking, wet autoclaving, and steaming of tissue sections yield satisfactory antigen retrieval results, comparative studies indicate that pressure cooking or pressure cooking in combination with microwave heating produces more uniform, efficient, consistent, and rapid immunostaining in some cases (Fig. 6.9). Pressure cooking with or without microwave heating provides temperatures higher than 100°C (superheating). Such temperatures can be obtained with the high-pressure microwave processor MicroMED URM (Sorisole, B G; Bergamo, Italy) (Suurmeijer and Boon, 1999). This apparatus provides controlled superheating under high pressure in the microwave processor. This processor has a maximal power output of 1,000W. The duration, temperature, and pressure can be adjusted with a touch screen personal computer. Microwave power and pressure are controlled through software. The pressure is regulated as a function of temperature, which facilitates heating of the antigen retrieval solution at a constant temperature higher than 100°C without bubbling. A glass dome designed to withstand pressure conditions rotates within the microwave cavity. The dome is provided with an automatic raising and lowering mechanism controlled by the personal computer. A fiberoptic sensor monitors the temperature of the antigen retrieval solution within the dome. The pressure in the glass dome is between 1,900 and 2,000 mbar. To obtain antigen retrieval, a plastic jar containing 250ml of 0.01 M citrate buffer (pH 6.0) is centrally placed in the dome within the microwave cavity. Different temperatures (ranging from 90–115°C), durations of heating (1–15 min), and pH values (2–10) can be tested to determine optimal parameters for retrieving a given antigen. For example, optimal immunostaining of Ki-67 antigen in malignant tumors using MIB-1 antibody was achieved at 115°C for 10 min at pH 6.0 (0.01 M sodium citrate buffer) (Suurmeijer and Boon, 1999). To my knowledge the use of this processor has not been reported by any other laboratory, perhaps because of its high price.

Antigen Retrieval at Low Temperature Heating treatment is one of the most important factors influencing the effectiveness of antigen retrieval on tissue sections. The heating of sections of the formalin-fixed and paraffin-embedded tissues at a high temperature (boiling) for 10–20 min is extensively used for retrieving many types of antigens. A variation of this method consists of heating at high temperature, followed by heating at moderately low temperature.

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Although antigen retrieval at high temperature yields excellent results in terms of nuclear immunostaining, cell morphology can be severely damaged. Such damage is either ignored or not readily visible at the resolution provided by the light microscope. Electron microscopy clearly shows this damage. The use of high-temperature heating is especially undesirable when studying fatty and fibrous tissues such as breast, skin, and gastrointestinal

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tract; morphological preservation of these tissues can be difficult at high temperatures. Also, sections of these tissues may be dislodged from slides (coated or uncoated) at high temperature. In some cases, regional staining of the section may also result from high temperature heating. The above-mentioned problems can be generally circumvented by using moderately low temperatures (60–80°C) for antigen retrieval. Such temperatures are also useful for antigen retrieval on archival sections that have been stored for years at room temperature on coated or uncoated slides. Dislodging of archival sections from slides during antigen retrieval is also minimized at moderately low temperatures. According to Biddolph and Jones (1999), these problems can be minimized by using low-temperature heating (60°C) in conjunction with boric acid as the antigen retrieval fluid. Loss of archival sections is especially serious because of the nonavailability of tissue blocks or if all the tissue has been used. If high temperature must be used for archival sections, loss of sections may be minimized by using an adhesive overlay on the sections before heating (Pateraki and Kontogeorgos, 1997). This approach requires further testing. Equal staining intensity is usually achieved at 80°C or at higher temperatures. Antigen retrieval can be obtained at 80°C in 10 mM citrate buffer using a water bath. A minor disadvantage to using moderately low temperature for antigen retrieval is the requirement of longer heating durations. The lower the temperature, the longer the duration of heating. As an average, heating at 80°C for 2hr is recommended. A wide range of heating durations at moderate temperature has been used in the published literature, which are listed below. Overnight heating (~ 15 hr) at 60°C has been used for retrieving muscle actin (HHF 35) and smooth muscle actin (CCG 7) (Igarashi et al., 1994). The same duration of heating but at 80°C was used in a conventional oven for retrieving estrogen receptor in the cervix tissue; Tris buffer (pH 9.0) was used as the antigen retrieval fluid (Koopal et al., 1998). Kawai et al. (1994) have reported that simple heating in a hot water bath at 90°C for 2hr was very effective for retrieving PCNA and p53 antigens (see Fig. 6.4). In this study, overnight heating at 60°C, using PBS or citrate buffer, also yielded good results. Heating at 90°C in a microwave oven for 15 min was also used for retrieving myosin heavy chain, using TUF antigen retrieval fluid; boiling was not allowed (Carson et al., 1998). Man and Tavassoli (1996) found that overnight heating at 70–80°C produced excellent staining of a number of antigens, including ER, PR, p53, and Ki-67. Moderately low temperature in conjunction with enzyme digestion has also been recently used for antigen retrieval; a few examples are cited below. The retrieval of Ki-67 antigen in surgical breast biopsy specimens has been achieved by treating the sections with 0.1% trypsin for 15 min at 37°C, followed by heating in 10 mM citrate buffer (pH 6.0) at 80°C for 2 hr in a water bath (Elias et al., 1999). In another study sections of breast cancer tissue were pretreated with 0.1% trypsin in PBS (preheated to 37°C) for 15 min, rinsed in deionized water, and then heated in l0 mM citrate buffer (pH 6.0) (preheated) in a water bath for 2hr for improved retrieval of estrogen receptor and Ki-67 antigen (Frost et al., 2000). A disadvantage of this combined treatment is the focal and sporadic digested appearance in the sections. These areas can be identified by the presence of inadequately stained nuclei by hemotoxylin. Moderate heating with or without enzymatic pretreatment is not the optimal method of immunodetection of all antigens. For example, more intense staining of antiapoptotic

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protein bcl-2 and progesterone in breast carcinoma is provided by heating in a microwave oven at full power than by moderate heating, although section loss is not uncommon with full-power heating. It is deduced from these and other studies that optimal retrieval of each antigen or antigen-antibody complex along with the tissue type requires specific intensity of temperature and its duration, and that the source of heat is not very important. Unfortunately, a universal temperature for retrieval of all antigen types in various tissues remains elusive.

Use of Heat for Staining Microwave heating accelerates the process of staining for light and electron microscopy, although this advantage is more useful for light microscopy because conventional staining is quite rapid for electron microscopy. Under microwave heating, the charged dye ions as well as the polar molecules and ions of the solvent, including water, are excited (Kok and Boon, 1990). The molecular movement generated by microwave heating may accelerate chemical reactions up to 1,200 times. The result is that heating speeds up diffusion of stains into the thick-tissue sections and their subsequent reaction and binding with the substrate. Generally, compared with conventional staining, staining in the microwave oven requires a much shorter staining duration and results in more intense staining, better contrast, and less nonspecific staining. In fact, the hours required for many conventional staining methods for light microscopy can be shortened into minutes. Several examples are listed below. One example is the Grimelius method for staining neuroendocrine granules in various tissues and tumors for light microscopy. The conventional procedure is completed in 3 hr, whereas the microwave method is accomplished in 3 min (Hopwood, 1992). Also, staining of melanin can be carried out with colloidal silver nitrate in 45 sec under microwave heating (Leong and Gilham, 1989a). Microwave heating is also effective in reducing the tissue staining time from 70min to 15min for localizing acid and neutral mucins with a modification of alcian blue periodic acid–Schiff stain (Matthews and Kelly, 1989). Microwave heat–stimulated staining of the brain tissue with the Rio-Hortego silver impregnation technique can be completed within 24 hr instead of the 7 days required by the conventional method (Marani et al., 1987). Satisfactory silver impregnation of cell bodies, axons and their terminals, and dendrites and their spines is obtained. Another example is the application of the Jones-Marres silver method for rapid staining of fungi in the brain tissue of immunocompromised patients (Boon et al., 1998). This procedure can be carried out using the MicroMED BASIC microwave lab station (Milestone, s.r.l., 24010 Sorisole, Italy). The lab station has software for reliable control of power, time, and temperature using infrared temperature control for no-touch temperature determination. It also has a 360-degree rotation carousel (no hot spots) and produces printouts of the temperature and power levels used during various microwave steps. Microwave heat can also be applied for rapid staining of frozen sections. Frozensection diagnosis plays an important role in the evaluation of the operability of the patient and in the examination of resection margins. The preparation for diagnosis, for example of signet-ring cell carcinoma in the peritoneum, can be accomplished in as brief a time as 30 sec by the modified periodic acid–Schiff’s (PAS) reaction facilitated by microwave heating

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(Dworak and Wittekind, 1992). This protocol can demonstrate even a small number of mucincontaining tumor cells surrounded by fibrous tissue in frozen sections (Fig. 6.10/Plate 3F). Microwave heat is also effective in staining SDS-polyacrylamide gels with Coomassie blue for visualizing as little as 5 ng of protein against a light blue background of the gel (Wong et al., 2000). This protocol is one of the most powerful methods in molecular biology for visualizing proteins. Rapid staining of frozen sections of human brain tissue that has been stored in 10% formalin (4% formaldehyde) for up to 10 years has also been reported (Feirabend and Ploeger, 1991). In this study, rapid staining was obtained in the microwave oven by using classic neuroanatomical staining methods such as Klüver-Barrera stain; originally Luxol fast blue step required up to 24 hr, whereas in the microwave oven this step needed only 15–60 min. Another application of microwave heating is rapid staining of plant tissues with dyes. For example, Safranin O can stain plant tissues in 45 min at 60°C in a microwave oven instead of the conventional 48 hr at room temperature (Schichnes et al., 1999). Microwave heat–assisted rapid fixation and double staining of the mouse fetal skeleton has also been carried out (Ilgaz et al., 1998). The staining was accomplished with a mixture of alcian blue and alizarin red S in 23 min in the microwave oven instead of 4 days at room temperature. The cartilage and bone are stained distinctly. Most staining methods require optimal temperatures and durations of staining. The optimal temperatures for most nonmetallic stains is 55–60°C, while for metallic stains it is 75–80°C (Suurmeijer et al., 1990). Some specific examples are given below: the Romanowsky-Giemsa method and the alcian blue technique at 55°C (Horobin and Boon, 1988), the Southgate mucicarmine procedure at 60°C, the Grimelius protocol at 75°C, the Grocott, Jones, and Fontana-Masson methods at 80°C (Kok and Boon, 1990), and the gold chloride (0.02%) at 74–98°C (Noyan et al., 2000). Some dye solutions, such as oil red O, can be used at boiling temperature.

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Table 6.1 indicates microwave power levels, the stain used, and the required temperature. Table 6.2 shows significantly reduced duration of staining under microwave heating compared with conventional staining conditions. However, many brands of microwave ovens are in use, and all vary in their performance, even at the same power level. Therefore, optimal stain concentration, temperature of staining, and durations of staining and rinsing will have to be determined for each type of new study. Also, care is required in interpreting the staining results because high temperatures tend to produce staining artifacts. Autostainers for histochemistry are available from the following sources: Leica Autostainer XL, Leica Instruments GmbH, Nussloch, Germany; Oticmax Rapid Microwave Histoprocessor Inc., 160 Shelton Road, Monroe, CT 06468.

Rapid Immunostaining of Frozen Sections Rapid immunohistochemical study of frozen sections is necessary for intraoperative diagnosis in some cases. Rapid immunostaining is also helpful in confirming or excluding tumor clearance in resection margins or in detecting micrometastases in sentinel lymph nodes in breast cancer patients. Two methods to immunostain frozen sections are the enhanced polymer one-step staining (EPOS) system and the EnVision system; both systems are detailed later.

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Using the horseradish peroxidase method and microwave heating, Ichihara et al. (1989) were also able to immunostain frozen sections for the intraoperative diagnosis of pancreatic cancer in 30min. Only four different antibodies were tested in this study. A shorter duration of 10min has also been used for immunostaining frozen sections with the EPOS system (Chilosi et al., 1994). The EPOS procedure is based on the chemical linking of primary antibodies and horseradish peroxidase to an inert polymer complex (dextran) (Bisgaad et al., 1993). This methodology has been employed for immunostaining of Ki-67, PCNA, cytokeratin, and leukocyte common antigens (Tsutsumi et al., 1995; Richter et al., 1999). The limitation of the standard EPOS system is that the primary antibodies are labeled and thus are commercially available only for limited range of antigens. In contrast to the EPOS system, a modification of the highly sensitive two-step irnmunohistochemical EnVision system allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min (Kämmerer et al., 2001). In this study 38 out of 45 antibodies tested showed specific staining. In fact, the modified EnVision procedure allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase molecules for detection. No attempt was made to block endogenous peroxidase, nor was any antigen retrieval pretreatment used. Because of the very short incubation durations, a humid chamber is not required to avoid evaporation of immunoreagents. A minor disadvantage of the modified EnVision system is that it requires primary antibody concentrations four- to tenfold higher than those used in the conventional immunohistochemical procedures. Another limitation of this modified method is that only two slides with two sections each can be processed at any one time.

Enhanced Polymer One-Step Staining Procedure Sections thick) of freshly frozen tissues are mounted on silane-coated slides and fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). The sections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibody for 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS, and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W) for ~ 1 min; during microwaving, the slides are cooled by a cold water bath (Werner et al., 1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for 10sec. They are rinsed in tap water and cover-slipped.

Modified EnVision Procedure Tissue specimens are snap-frozen in liquid nitrogen for 30 sec immediately after removal and then transferred to a cryostat (Kammerer et al., 2001). Serial frozen sections of thickness are cut and placed on silane-coated slides. They are air-dried for 30 sec, fixed in acetone for 1 min at room temperature (22°C), and air-dried at 22°C (Fig. 6.11). The sections are incubated with primary antibody in the antibody diluent (Dako) for 3 min by placing the slide horizontally on a hot plate at 37°C. (All incubation steps are carried out by placing the slide horizontally on the hot plate at 37°C.) Following a brief rinse in TBS, the sections are incubated with the goat-anti-mouse EnVision-HRP-enzyme conjugate for 3min at 37°C.

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The sections are rinsed in TBS, and then exposed to DAB+ chromogen (Dako) for a few minutes as the substrate for the EnVision-HRP-enzyme. The sections are washed by shaking the slide rapidly under tap water for 10 sec. The excess fluid is removed from the slide with a paper towel. The slide is dipped in distilled water, counterstained with Meyer’s hematoxylin for 15 sec, and then rinsed in hot tap water at 42°C for 30 sec.

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Hazards and Precautions in the Use of Microwave Ovens The causes of most hazards encountered in using a microwave oven are straightforward and can be avoided by taking necessary precautions. Higher-power settings and longer durations of heating than optimal for a given study should be avoided. Because overheating is not uncommon, the time setting should be checked. The fluid contents of the container heat faster than the container. In fact, the fluid contents of the container heat so fast that the container can still be cool (Marani, 1998). Even after the container has been removed from the oven, it will become hotter for a period of time. Changes in the size, shape, and nature of the container and its position in the microwave oven significantly change the temperature of the container fluid. Furthermore, changes of these factors will change the temperature of the container fluid even if the volume of the container contents remains unchanged. Overheating the microwave oven tends to result in boiling or excessively rapid evaporation of fluids such as ethanol used for dehydration, formaldehyde employed for fixation, and the antigen retrieval fluid. As a result, flammable and/or toxic materials are released in the microwave oven. Even without overheating, vapors are produced because containers are kept open in the oven to prevent pressurization. Transparent microwave containers should be used, fluid volumes should be ~100ml. Microwave ovens with attached efficient extractor fans are commercially available, as are microwave ovens with temperature probes. To avoid possible exposure to toxic vapors, the face should be turned away when the oven door is opened (Horobin and Fleming, 1990). The oven door should not be opened or closed to turn the microwave power on and off. When using Pelco 3440 MAX laboratory microwave oven (Ted Pella, Redding, CA), areas of high microwave flux should be checked, using a Pelco 3,614 microwave bulb array (Ted Pella) (Fig. 6.12). Specimens should not be placed in areas indicated by illuminated bulbs. Vials containing the specimens should be placed in a water bath (50 ml) that has been preheated to the required temperature. The temperature should be regulated by placing a microwave temperature probe into a vial of the same solution that is present in the specimen vial. The built-in temperature probe displays the temperature on the oven front panel. The wire that attaches the probe to the oven should be submerged in the water to decrease the antennae effect (Schichnes et al., 1999). An additional 400ml of static water load should be placed in the oven at an optimal position determined with the microwave bulb array. This water is changed between every step. Pelco BioWave microsystem is the latest advancement in microwave heating technology. It is equipped with vacuum cycling (down to 1 torr), variable wattages, and a precision temperature probe; it can accommodate Pelco coldspot connected to the Pelco load cooler and thus eliminate hot and cold spots during processing. The system can be used for both light and electron microscopy. The vacuum chamber is most helpful during fixation and infiltration of tissue specimens. The following specific steps must be taken while using a microwave oven for antigen retrieval (Marani, 1998). 1. Test microwave leakage with a microwave detector with a low sensitivity range. 2. Place the oven in an efficient fumehood. 3. Wear gloves while using your hands inside an oven.

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4. Predetermine the maximum power level of the oven to be used. 5. Do not place a container with a closed lid in the oven. 6. Do not use high temperature settings unless absolutely necessary. 7. Predetermine the heating time, and check the time setting. 8. Check the actual temperature attained by the specimen. 9. Predetermine the number of specimens to be heated. 10. Predetermine the exact position of the specimen in the oven during heating. 11. Predetermine the amount of a water load and its place in the oven. 12. Find out the extent of hot spots in the oven. 13. Use Teflon containers with thick walls in the oven. Plastic containers can also be used. 14. Do not use metals or foils in the oven. 15. Contrary to some published reports, pencil-written materials can be used in the oven.

Limitations of Microwave Heating In spite of the overwhelming advantages of microwave heating, some real and possible limitations are described. Background staining may occur with some antibodies, particularly when the heating is prolonged. This problem can be avoided by determining the optimal temperature and time of heating by trial and error. Although antigen specificity

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for the monoclonal antibody is maintained after microwave treatment, the possibility of altered immunostaining should not be disregarded whenever new or previously untested antibodies are used. The results of such studies should be compared with those obtained using frozen section immunohistochemistry. In some cases, microwave heating may damage nuclear morphological details, including mitotic figures. This problem may lead to difficulty in identifying cells accurately, which is important in diagnostic studies. Both mitotic figures and morphology, for example, are important in distinguishing a malignant lymphoid infiltrate within a mixed cell population (Hunt et al., 1996). In such studies, it is desirable to use a heating method other than microwaves and accept slightly lower immunostaining enhancement. Although the exact knowledge of molecular changes responsible for impairment of nuclear morphology caused by microwave heating is lacking, it may be possible that this treatment causes some structural damage to intracellular macromolecules, resulting in an increase in the number of osmotically active moieties within the nuclear compartment, thus attracting water and causing nuclear swelling (Hunt et al., 1996). Such swelling would blur mitotic figures, leading to a less accurate count of them. Some other limitations and their avoidance are described below. With violent boiling and extensive evaporation of the retrieval fluid in which the sections are immersed, the sections should be monitored to avoid drying and damage. To obviate this problem, microwave heating must be performed in repeated bursts; the plastic jars must be refilled following each cycle or a large reservoir of retrieval fluid or distilled water must be placed in the oven. To avoid inconsistent results, plastic jars containing the slides should always be placed every time in the same location in the microwave oven. The number of slides and jars should be constant every time a microwave oven is used, even when this entails inserting blank slides into the jar (Gown et al., 1993). Tissue sections should be placed toward one end of the slide (lower side of the slide while placing it in the jar) to ensure continuous immersion in the epitope retrieval fluid during microwave heating. Uneven distribution of microwaves within the oven results in hot and cold spots (see pages 102–103). This problem can be avoided by placing a 500-ml water load in the rear of the oven and by using a turntable during the process of heating (Panasonic model NN 5652, 800 W). Only a limited number of slides can be accommodated in the microwave oven. Tissue section detachment from the glass slide may occur during heating, especially with tissues containing prominent fibrous elements (Cuevas et al., 1994). If this problem is encountered, the surface of the slide can be made adhesive for sections by coating it with poly-L-lysine or 3-aminopropyl-triethoxysilane or, still better, by using electrically charged glass slides. Another problem is that microwave ovens have the inherent disadvantage of decreased power generation with use. Thus, no two ovens in use will have the same heating characteristics. This limitation is an obstacle in standardizing antigen unmasking methods. Microwave heating in some cases is not desirable. This method, for example, causes complete loss of estrogen receptor immunoreactivity, even when monoclonal antibody H222 is used (Gown et al., 1993; Leong, 1996). In such cases, alternate procedures, such as enzyme digestion alone or followed by microwave heating, can be used. Similarly, another steroid hormone receptor androgen shows stronger immunostaining with autoclaving than that using microwave heating (see Fig. 6.13) (Ehara et al., 1996). Another example is insulin, which shows diminished immunoreactivity after microwaving in citrate

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buffer (pH 6.0) (Tornehave et al., 2000). In such cases alternative antigen retrieval fluids and heating methods are required. Contrary to some reports, microwave heating in some cases does not abolish contaminating immunostaining during the consecutive detection of two or more types of antigens within the same section. This problem is especially common when double immunolabeling with antibodies of the same species and isotope is used. Recently it was demonstrated that microwave heating did not completely abolish contaminating staining when cytoplasmic and nuclear antigens in proliferating cells were labeled in cryostat and paraffin sections, with primary monoclonal antibodies from the same species and the same isotope being used (Bauer et al., 2001). However, such contaminating staining can be avoided in some cases with the use of microwave heating. Lan et al. (1995) have reported blocking of antibody cross-reactivity in multiple immunoenzyme staining and retrieving antigens with microwave heating. In summary, although the microwave heating method is highly effective for detecting a large number of tissue-bound antigens which otherwise may remain masked, primarily due to fixation with formaldehyde, certain antigens show reduced immunoreactivity following microwave heating. It should also be noted that epitope retrieval with microwave heating or other methods can unmask cross-reactivities that can be very difficult to deal with. Therefore, the use of retrieval methods for immunostaining creates the necessity for increased vigilance in the selection and interpretation of controls, both negative and positive.

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WET AUTOCLAVE METHOD Wet (hydrated) autoclave pretreatment as an alternative to microwave heating was introduced by Shin et al. (1991) for unmasking tau protein on sections of brain tissue fixed with formalin and embedded in paraffin. This methodology was later modified by Bànkfalvi et al. (1994) for diagnostic antigen retrieval. The justification for employing autoclaving is that microwave heating is less desirable than equivalent autoclaving for the retrieval of certain antigen types. For example, microwave heating may damage nuclear morphological details, including mitotic figures. Some evidence indicates that cell morphology is preserved better with autoclaving than with microwaving. It has been suggested that autoclave heating does not cause loss of sections. The retrieval of certain types of epitopes requires temperatures higher than 100°C, which is provided by an autoclave (120°C). It is thought that damage by the super-high temperature to cell morphology is comparatively less in some cases. The super-high temperature has been reported to result in stronger immunostaining of steroid hormone receptors than with that obtained with microwave heating (100°C for two pulses of 5min each) (Fig. 6.13) (Ehara et al., 1996). Also, compared with microwaving, autoclaving produces superior immunostaining of progesterone (Mote et al., 1997). In addition, autoclaving yields better immunostaining of U2-OS cell nuclei for retinoblastoma susceptibility gene product (RB) compared with that achieved by using microwave heating (Tsuji et al., 1998). Furthermore, immunostaining of fibronectin epithelial nucleus in oral mucosa was not present on frozen sections, but such staining was achieved after autoclaving (Mighell et al., 1995). However in some cases, autoclaving is undesirable. This is exemplified by the abolition of immunostaining of calcineurin in the CNS neurons after autoclaving (Usuda et al., 1996). Autoclave pretreatment (100°C) may damage cell morphology of fatty tissues or tissues containing areas of fatty tissues (e.g., breast tissue). The extent of damage can be minimized by mounting the sections on protein-coated glass slides that have been allowed to dry for 48–72 hr before autoclaving (personal communication, 1999, K. W. Schmid). In addition to its effectiveness in antigen retrieval in the cases above, an autoclave has the advantage of accommodating a much larger number of slides than does a microwave oven. Several hundred slides can be simultaneously processed in an autoclave, eliminating possible immunostaining variations when small batches of slides are microwave-heated at different times. Moreover, antigen retrieval fluid is not lost during heating in an autoclave. In contrast, in a microwave oven the jar containing the slides must be refilled after each heating cycle. However, this tedious exercise can be avoided by using a large reservoir of fluid to minimize the possible deleterious effects of boiling or drying of the sections. A drawback of autoclaving is the high cost of the equipment. The use of a pressure cooker is a cheaper alternative for a smaller number of sections.

Procedure 1 Tissues are fixed with formalin for 18 hr to 4 weeks and then embedded in paraffin. Sections are mounted onto superfrost or poly-L-lysine–coated glass slides, dried in an oven for 1 hr at 60°C, and deparaffinized with three changes of xylene. This is followed by rehydration through a series of descending concentrations of ethanol. The slides are placed

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in plastic Coplin jars containing 0.01 M sodium citrate buffer (pH 6.0), which are heated in an autoclave for 5–10 min at 120°C. The slides are allowed to cool down to room temperature for 20–30 min and then briefly rinsed in 0.05 M Tris-HCl buffer (pH 7.4) or 0.1 M PBS. Blocking of endogenous peroxidase activity is accomplished by immersing the sections for 30 min in a solution of 0.3% in distilled water, and then rinsing in PBS. If needed, background staining can be blocked by treating the sections for 10–30 min at room temperature with normal serum from the species supplying the second antibody at a dilution of 1:5 to 1:20 in PBS. The sections are incubated overnight in a humidified chamber at 4°C in the primary antibody at an appropriate dilution. They are rinsed three times for 5 min each in PBS, further processed by using avidin-biotin complex (Vectastin, Vector Labs, Burlingame, CA), followed by DAB as the chromogen. Counterstaining of the nuclei is accomplished with hematoxylin or methyl green. As a negative control, irrelevant antibody UPC10 (Cappell, Organon Teknika, West Chester, PA) can be used or primary antibody can be omitted. The sections are mounted in an appropriate mountant.

Procedure 2 The following hydrated autoclave method can be employed for immunohistochemical detection of molecules in both cultured cell and tissue specimens. The method was used, for example, to localize androgen receptor in cultured LNCaP cells (derived from prostatic carcinoma metastasized to lymph node) and biopsy specimens from patients with prostatic carcinoma (Ehara et al., 1996). After being removed from the culture medium, the cells on plastic cover slips are fixed with 10% formalin for 10 min at 20°C. Tissue specimens are fixed for 1–2 days and embedded in paraffin. Sections are cut, mounted on glass slides, and heated in an oven for 1 hr at 42°C to promote adherence to the slide. After deparaffinizing and rehydration, the sections are subjected to epitope retrieval treatment as follows. The slides are placed in metal slide racks and immersed in a beaker filled with 0.01 M citrate buffer (pH 6.0). The beaker is loosely covered with a sheet of aluminum foil and autoclaved for 15 min at 120°C. After cooling to room temperature, the autoclave lid is taken off. The sections are treated with 3% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. As a blocking solution, 10% normal goat serum is used for 10 min. The sections are reacted with the primary antibody at an appropriate dilution at 4°C in a moist chamber. After being washed with 0.075% Brij 35 (Sigma Chemical Co., St. Louis, MO) in PBS three times, the sections are treated with an appropriately prediluted antibody for ~10 min in a moist chamber. After washing, the sections are reacted with the prediluted HRP-labeled streptavidin for ~5 min. The sections are washed, and the HRP site is visualized with DAB, hydrogen peroxide, cobalt, and nickel, without counterstaining. As a negative control, the sections are reacted with normal mouse serum, normal IgG, or normal rabbit serum in place of the specific antibodies after autoclaving (see Fig. 6.13).

ULTRASOUND TREATMENT Ultrasound (sonication) converts AC line voltage to 20-kHz high-frequency electrical energy, which is fed to a converter where, in turn, it is converted to mechanical vibrations.

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The main part of the converter is a lead zirconate titanate electrostrictive element that expands and contracts when subjected to alternating voltage (Portiansky and Gimeno, 1996). The converter vibrates in a longitudinal direction and conveys this motion to the horn tip immersed in the solution, resulting in the implosion of microscopic cavities in the solution. The implosion causes the molecules in the solution to become exceedingly agitated. This phenomenon is explained below. Some information is available on the mechanisms responsible for the direct or indirect effects exerted by ultrasound on antigen retrieval. Considerable heat is generated during ultrasound exposure, but the heat dissipates very quickly. Very rapid heat loss has misled some workers to state that “ultrasound generates a mild increment in temperature” (Portiansky and Gimeno, 1996). Ultrasound waves consist of cycles of compression and expansion. Compression cycles exert a positive pressure on the liquid, pushing the molecules together, whereas expansion cycles exert a negative pressure, pulling the molecules away from each other. The tensile strength of solutions is reduced by gas trapped in the crevices of small solid particles in the solution. When a gas-filled crevice is exposed to a negative pressure cycle from a sound wave, the reduced pressure makes the gas in the crevice expand until a small bubble is released into the solution, initiating cavitation. A negative pressure of only a few atmospheres will form bubbles. The bubbles ( in diameter) implode violently in less than a microsecond, intensely heating their contents (Suslick, 1989). Thus, during the expansion cycle a sound wave of sufficient intensity can generate cavities in the solution. Ultrasound treatment causes enormous molecular agitation (turbulence), heat, and pressure of imploding cavities. Such agitation not only initiates but also accelerates both biochemical and physical reactions. In other words, effects of ultrasound involve processes that create, enlarge, and implode gaseous and vaporous cavities in a solution. The implosion of cavities also sends shock waves through the solution. This extreme condition generated by cavitation can induce reactivity between cellular proteins and the antigen retrieval solution (e.g., sodium citrate). Mechanical vibrations and high temperatures may extract tissue-bound calcium ions, accelerating the chelating effect of citrate. This suggestion is reinforced by the evidence that ultrasound hastens calcium chelation and bone decalcification (Thorpe et al., 1972; Page et al., 1990). Chelation of calcium may result in epitope retrieval (Morgan et al., 1994). It is known that ultrasound can break or disrupt cells and tissues. Mechanical vibrations generated by ultrasound can induce structural changes in the tissue sections, breaking the formalin-introduced protein crosslinks and thus facilitating the accessibility of antigens to antibodies. Ultrasound can also unfold or “crack” protein molecules into smaller fragments, exposing the epitopes. The effectiveness of ultrasound treatment in epitope retrieval has been compared with that achieved with microwave heating or pressure cooker alone (Portiansky and Gimeno, 1996). It was shown that ultrasound was more effective in immunostaining prostatic basal cell structural cytokeratins. The capability of microwave heating for epitope retrieval has also been compared with that of ultrasound in combination with microwave heating (Brynes et al., 1997). The latter approach resulted in stronger immunostaining with lower nonspecific background staining of cyclin Dl bcl-1 nucleoprotein in mantle cell lymphoma specimens. It should be noted that raising the temperature beyond a certain level in the presence or absence of ultrasound does not improve epitope retrieval and in addition results in excessive

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background staining. A limitation of ultrasound heating is that it is difficult to reproduce between laboratories, as the exact parameters regarding the intensity at the acoustic frequency are difficult to set precisely. The results of the studies carried out by Hammoud and Van Noorden (2000) using ultrasonication do not agree with those reported by Portiansky and Gimeno (1996). The former authors do not recommend using this technique in a routine histopathology laboratory. Miller et al. (2000) also report that the results obtained with ultrasonic treatment are inferior to those achieved with a pressure cooker. The discrepancy between the results obtained with these two teams and other workers may be due to the difference in the type of antigens ultrasonicated in various laboratories under different processing conditions. For example, Portiansky and Gimeno (1996) used an ultrasonic cell disrupter with an output of 40 W, whereas Hammoud and Van Noorden (2000) employed an ultrasonic cleaning bath having an output of 80 W. Also, various studies used tissues fixed for different durations. Tissue section adhesion to slides has also been reported to be a problem during ultrasonication. In spite of the lack of agreement on the usefulness of ultrasonic treatment, this method requires very short duration (~40 sec) for antigen retrieval. However, the usefulness of ultrasonic treatment requires additional substantiation.

Procedure Tissues are fixed with 10% formalin for 7–10 days and embedded in paraffin (Portiansky and Gimeno, 1996). Sections about thick are mounted on glass slides coated with poly-L-lysine and deparaffinized with xylene. They are incubated with 0.03% methanolic hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. Following dehydration with graded ethanol, they are rinsed in deionized water and then in PBS. The glass slides containing these sections are vertically oriented in the lateral walls of a 75×95-mm glass dish and completely covered with 10 mM citrate buffer (pH 6). The tip of the cell is disrupted (Branson Ultrasonics model 250), set to continuous mode, and immersed 3 cm in the citrate buffer in the center of the dish. After incubation in the primary antibody (appropriately diluted), the avidin-biotin complex (ABC) is used as the detection system. In control sections, the primary antibody is replaced with normal mouse serum.

NONHEATING METHODS

Detergents Antigen retrieval using heat-based methods is not being widely used for cell cultures and cryosections fixed with an aldehyde. The immunolabeling efficiency of such specimens can be improved by using a chemical antigen retrieval protocol. This protocol consists of permeabilizing the specimens with Triton X-100, followed by treating with sodium dodecyl sulfate (SDS). This permeabilization/denaturation treatment is applied after fixation and prior to incubation with the primary antibody. SDS is the most commonly used denaturing agent for gel electrophoresis. The application of SDS in epitope retrieval is based on the observation that after treatment with this reagent, protein bands appear in gel electrophoresis, but

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such results with similar proteins without SDS treatment using immunocytochemistry are not visible. Being a protein denaturant, SDS application may result in bands after staining with Coomassie blue, but it itself is not a stain. Therefore it should not be referred to as a positive or negative stain. SDS disrupts noncovalent interactions between subunits of a protein, so if a protein has two subunits, two bands will appear. In the absence of SDS, only one band will appear. This reagent and mercaptoethanol reduce protein subunits that are disulfide bonded. This property of SDS may be responsible for protein denaturation. It should be noted that SDS also permeabilizes cells for antibody access to intracellular epitopes. Triton X-100 and digitonin are also used to permeabilize the cell membrane allowing antibody penetration. Triton X-100 permeabilization of formaldehyde-fixed cells allows antibodies better access to their epitopes than does digitonin treatment. Digitonin or saponin binds to cholesterol within membranes, creating digitonin-cholesterol complexes and pores in the membrane. The pores are sufficiently large to allow antibody penetration. On the other hand, Triton X-100 is a stronger detergent and dissolves most of the membrane lipids. As a result this detergent increases the accessibility of antibodies to cell compartments that are not permeabilized with digitonin (Hannah et al., 1998). A limitation of Triton X-100 is that it may extract certain antigens even from fixed cells. Thus, false-negative staining of antigens, especially membrane antigens, of the cells treated with a strong detergent can occur because of antigen extraction. However, it should be noted that not all membranes of formaldehyde-fixed cells are impermeable to antibodies without permeabilization. The permeabilization/denaturation method has been successfully used for immunolabeling of in human neutrophils and MRC-5 cells (Robinson and Vandré, 2001). The method has also been effective in labeling MDCK cells in conjunction with indirect immunofluorescence (Brown et al., 1996). It should be noted, however, that some type of antigens remain masked, while other types may be adversely affected by SDS treatment. Still other antigens (e.g., aquaporins and brush border gp330) remain unaffected by SDS treatment (Brown et al., 1996). An example of an antigen whose staining is negatively affected by SDS treatment is in the Golgi complex; this occurs with the anti-AE1 anion exchanger antibody (Brown et al., 1996). Therefore, the usefulness of the SDS treatment should be assessed in each case. Caution is also required to prevent drying out of the specimens during incubation steps because they become hydrophobic with SDS treatment.

Procedures Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion (Brown et al., 1996). Tissue slices are further fixed overnight at 4°C with the same fixative and stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30% sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embedding medium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sections about thick are cut at a chamber temperature of –25°C, collected on Fisher Superfrost Plus charged slides, and stored at –20°C until use. The sections are brought to room temperature, and a wax pen (PAP pen, Kiyota International) is used to trace a hydrophobic circle around each section. They are rehydrated by immersion in PBS for 5 min; most of the PBS is removed from the slide with a tissue paper and the sections are then covered with drops of SDS solution (1% SDS in

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PBS). The drops are confined to areas where the sections are encompassed by the wax circles. After the slides have been treated horizontally for 5 min at room temperature, the slide is immersed in PBS in a Coplin jar to remove the SDS. The control slide not exposed to SDS is washed in a separate jar to avoid any contact with SDS. The slides are thoroughly washed three times for 5 min each with PBS, completely removing the SDS; otherwise, residual SDS will denature the antibodies subsequently applied to the sections. While the slide is horizontal, excess PBS from the areas outside the wax circles is removed. The sections become hydrophobic after SDS treatment, so care must be taken to prevent them from drying. The aliquot of primary antibody should be already in the pipette, so that it can be applied to the sections immediately after the residual PBS has been removed. The sections can be incubated in the primary antibody for 1–2 hr at room temperature, followed by two washes for 5 min each in high-salt PBS (containing 2.7% NaCl instead of 0.9% NaCl). This PBS minimizes nonspecific binding of antibodies to the tissue. After being washed for 5 min in normal PBS, the sections are incubated in the secondary antibody (goat antirabbit IgG conjugated to fluorescein isothiocyanate, FITC) for 1 hr. This is followed by washing in normal PBS, then mounting of sections in the medium of choice (Fig. 6.14). A second nonheating epitope retrieval method involves the use of sodium hydroxidemethanol solution. This solution was used successfully for epitope retrieval in sections of formalin-fixed, acid-decalcified human temporal bone embedded in celloidin (Shi et al., 1991). This solution is prepared by adding 50–100 g of NaOH to 500 ml of methanol in a brown bottle and mixing vigorously. The solution can be stored for 1–2 weeks at room temperature; it is also available commercially (BioGenex, San Ramon, CA). The clear, saturated solution is diluted 1:3 with methanol before use. A wider application of this solution is awaited. Another reagent used to unmask epitopes by denaturing antigens is guanidine hydrochloride (GdnHCl) which is freely soluble in water and alcohol; its

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aqueous solution has neutral pH. It was used for retrieving masked or hidden intracellular protein in scrapie-infected cultured cells (Taraboulus et al., 1990). A modified version of this protocol was employed for localizing different epitopes in BHK-21 cells fixed with paraformaldehyde containing small amounts of glutaraldehyde (Peränen et al., 1993). These epitopes were undetectable without denaturing the antigens with GdnHCl, using immunofluorescence microscopy. The advantage of denaturation of antigen complexes using GgnHCl is that it allows the use of glutaraldehyde, a more potent protein cross-linker, which better preserves protein cell structure. This means that low concentrations of this dialdehyde do not interfere with the epitope retrieval property of GgnHCl. The use of glutaraldehyde widens the applications of this denaturing agent. Both monoclonal and polyclonal antibodies can be used in conjunction with GgnHCl. Guanidine hydrochloride is especially useful with antibodies known to react only with denatured antigens. This reagent also permeabilizes cells. A limitation is that GgnHCl tends to eliminate the antigenicity of certain intracellular structures such as microtubules. However, microtubule loss can be prevented by using low concentrations of glutaraldehyde during fixation with paraformaldehyde (Peränen et al., 1993).

Proteolytic Enzyme Digestion A variety of proteolytic predigestions have been employed for unmasking epitopes that had become inaccessible as a result of crosslinking during aldehyde fixation. The digestive treatments have been carried out most commonly with trypsin, pepsin, proteinase K, or pronase (their concentrations are given later) prior to immunostaining. Detailed comparative studies on the effects of these four enzymes on epitope unmasking demonstrate that while the results did not differ significantly among themselves, their effects did differ, depending on the tissue and the antibody used (Hazelbag et al., 1995). Other factors affecting such results include the duration of digestion, pH, temperature, and length of fixation. The mechanism responsible for antigen retrieval by enzymatic digestion is breakdown of protein crosslinks formed during formalin fixation. It is likely that enzyme treatment digests surface binding proteins, exposing the masked antigenic sites for antibody binding. This idea is supported by evidence that the duration of enzymatic digestion required for epitope retrieval is proportional to the length of formaldehyde fixation. It is also known that overdigestion leads to damage, not only to cell morphology but also to immunoreactivity. Enzymatic digestion is preferred over microwave heating for antigen retrieval in a few cases. Even multiple enzymatic digestion is required to retrieve certain antigens in a specific tissue. As an example, it has been reported that the monoclonal antibody RCC is most effective in the staining of clear cell carcinomas and papillary carcinomas in renal neoplasms when sections are pretreated with a three-step enzymatic digestion method: 0.12% trypsin in Tris-buffered saline (TBS), 0.01% pronase in TBS, and 0.1% pepsin in 0.1 N HC1. Results were inconsistent with heat-induced epitope retrieval techniques. However, trypsin is used most commonly, which catalyzes the hydrolysis of orginyl and lysyl peptide bonds. Trypsin usually is used at a concentration of 0.1% in 0.05 M Tris/HCl buffer (pH 7.8) containing 0.1% for 20–40min at 37°C. The addition of is essential

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for controlling digestion and reducing the production of occasional white flocculation (Macintyre, 2001). Only freshly prepared solutions of these enzymes should be used, as enzyme activity decreases with age. Also, these solutions should be prewarmed to the required temperature to ensure consistent results. Proteolysis does have certain limitations. Some antigens are susceptible to enzyme digestion. In some cases insufficient unmasking can result in poor or false-negative results, while excessive digestion may adversely affect cytomorphological features and cause increased background staining and detachment of tissue sections from the slide. It is known that proteolysis is a potent treatment. Because the cleavage of the protein molecule by proteolytic enzymes is mostly nonspecific, these reagents may alter the epitopes. In other words, peptide bond cleavage by these treatments is largely nonspecific. Therefore, these procedures are not the preferred treatments. Nevertheless, enzymatic digestion is useful for a limited panel of antibodies. If needed, enzymatic pretreatments can be applied preceded by microwave heating (Dookhan et al., 1993).

Procedure Slides with tissue sections are treated with 0.1% trypsin solution containing 0.1% (pH 7.4) for 15 min at 37°C, with 0.4% pepsin solution containing 0.01 MHC1 for 20 min at 37°C, or with 0.025% pronase E solution containing 0.05 M Tris-HCl (pH 7.6) for 15 min at the same temperature (Hazelbag et al., 1995). These are average concentrations and durations, which should be adjusted according to the tissue and antigen type and the duration of fixation. Prolonged fixation requires longer proteolysis to unmask the epitopes. Excessive proteolysis results in decreased immunostaining. If loss of the sections during proteolysis is a problem, the slide can be coated with a 3% solution of casein white glue and dried overnight before the sections are placed on it.

Enzyme Digestion and Relatively Low Temperature (80°C)–Assisted Antigen Retrieval High-temperature microwave heating is currently the most widely used antigen retrieval method. This approach has significantly improved the detection of a wide variety of antigens. In some studies low-temperature (80°C) antigen retrieval is more effective than that obtained with high temperature. This is exemplified by restoration of estrogen and progesterone immunoreactivity (Elias and Margiotta, 1997). Recently, it was reported that sequential use of trypsin digestion and low-temperature heating (80°C) was more effective than high-temperature retrieval of Ki-67 antigen in breast tumors, using MIB-1 antibody (Elias et al., 1999). Another reported advantage of the former approach is that it causes the least amount of section loss during heating; sections of tissues with a high fat content may be dislodged from the slide at a high temperature. Surgical breast biopsy specimens are first fixed with neutral buffered formalin (4%) for 4–6 hr, followed by zinc-formalin for 2 hr. Paraffin sections ( thick) are placed on silane-coated slides, dried on a slide warmer (60°C) for 1 hr and then in an oven (60°C) for an additional 1 hr. Deparaffinized sections are digested with 0.1% trypsin in PBS at 37°C for 15 min. The sections are placed in 10 mM citrate buffer (pH 6.0) and transferred into a water bath (80° or 90°C) for 2 hr. After a 20-min cooling period, the sections are rinsed

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with PBS and then incubated overnight at 4°C in the MIB-I antibody (Immunotech, Westbrook, ME), diluted 1:50 with PBS. An automatic immunostainer (Cadenza, Shandon Scientific Inc., Pittsburgh, PA) can be used to accomplish staining. The Supersensitive Streptavidin-AP detection kit (BioGenex, San Ramon, CA) is used according to manufacturer’s directions. The final color reaction is developed with a fast red substrate (BioGenex), followed by mild hematoxylin counterstaining to avoid masking weak immunostained nuclei. Slides are coverslipped with Crystal Mount (Biomedia Corporation, Foster City, CA).

COMPARISON OF ANTIGEN RETRIEVAL METHODS: A SUMMARY The following recent comparative studies demonstrate that no single antigen retrieval method is optimal for all types of antigens. 1. Immunostaining of and isoforms of calcineurin in the human brain employing CAN-2 and CAN-3, respectively, was compared between microwave heating (in 10 mM sodium citrate at pH 6.0 for 10 min) and autoclaving at 120°C for 20 min (Usuda et al., 1996). The former approach was the most effective for intensification of the immunoreaction. 2. Compared to enzyme digestion methods, microwave heating demonstrated more intense immunoreactivity of estrogen and progesterone in breast cancer tissues fixed with methacarn (60% methanol, 30% chloroform, and 10% acetic acid) (Oyaizu et al., 1996). 3. Compared with trypsin digestion, microwave heating produced more consistent results and was effective over a greater range of fixation tissues in the case of immunoglobulin light chain in tonsil tissue (Ashton-Key et al., 1996). 4. Compared with pepsin predigestion, microwave heating markedly enhanced the staining of aberrant p53 antigen with Pab 1801-D07 antibody cocktail in paraffin or frozen sections in adenocarcinoma of the lung (Resnick et al., 1995). 5. Compared with microwave heating (three times for 5 min each at 100°C), hydrated autoclaving (5 min at 121°C) yielded stronger immunostaining of bcl-2 using bcl-2, 124 antibody (Umemura et al., 1995). 6. Compared with nonhydrated autoclaving, hydrated autoclaving produced stronger immunostaining of tau (a microtubule-associated protein) using anti-PHF/tau and antihuman tau (Shin et al., 1991). 7. Compared with microwave heating, heating on a hot plate yielded better immunostaining of IgG using antihuman IgG on epoxy thin sections for electron microscopy (Stirling and Graff, 1995). 8. Compared to microwave heating, superheating (120–122°C) for 1–2 minutes in a pressure cooker gave better immunostaining of IgD (Norton et al., 1994). 9. Immunostaining of a wide variety of biopsies was studied using different antigen retrieval fluids and heating and digesting systems (Pileri et al., 1997). This study showed the superiority of pressure cooking and EDTA over other methods, including microwave heating and proteolytic treatment.

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10. Ultrasound treatment was compared with microwave heating and pressure cooking;

11.

12.

13.

14.

15.

16.

17. 18.

the former treatment was claimed to be quantitatively and statistically superior for the immunostaining of prostatic basal cell structural cytokeratins using monoclonal antibody K8.12 (Portiansky and Gimeno, 1996). Compared with trypsinization–microwave heating, microwave heating– trypsinization demonstrated optimal immunostaining of Ki-67 using monoclonal antibody MIB-1 (Szekeres et al., 1995). Immunostaining using a panel of 21 antibodies was compared by employing microwave heating, microwave–pressure cooking, autoclave, and steamer (Taylor et al., 1996b). These methods yield similar intensities of staining provided the durations of heating are appropriately adjusted. Immunostaining of cytokeratin 18 in normal and neoplastic hepatocytes using antibody CK 18 was compared by employing microwave heating (15 min), autoclaving (10 min), pressurized boiling (1min), and simple boiling (15 min) in 10 mM citrate buffer (pH 6.0) (Xiao et al., 1996). No difference was found in the degree of immunostaining with light and electron microscopy. Compared to microwave heating, digestion with proteinase K for 2–4 min at room temperature yielded better retrieval of cytokeratins in mouse tissues using monoclonal antibodies (e.g., AE1, AE3) generated against human cytokeratins (Martin et al., 2001). Immunostaining of a number of proteins between microwave heating at 100°C of 20 min and boiling on a conventional hot plate. No difference was observed in the results of the two methods (Varma et al., 1999). Antigens bcl-2, CD3, and CD79a in tonsil tissue embedded in methyl methacrylate show superior immunostaining with trypsin followed by superheating at 121°C in a pressure cooker compared with that obtained with microwave heating only (Hand and Church 1998). Among the three antigen retrieval methods, hydrated autoclaving, microwave heating, and simple heating, simple heating overnight at 60°C was most effective for smooth muscle actin labeling (Igarashi et al., 1994). More intense and widely distributed staining of cytokeratins was observed with protease digestion than with microwave heating in benign lesions in the prostate using mouse monoclonal antibody (Googe et al., 1997).

Chapter 7

Antigen Retrieval on Resin Sections

Most commonly, antigen retrieval involves heating sections of paraffin-embedded tissues prior to light microscopy. However, antigen retrieval can also be accomplished on sections of resin-embedded tissues (Fig. 7.1). Tissues embedded in a resin show superior preservation of cellular details compared with those embedded in paraffin. Moreover, resin sections permit high resolutions to be obtained. In addition, semithin or thin (8–100 nm) sections can be obtained from resin-embedded tissues, allowing correlative studies using light and electron microscope, respectively (see Fig. 1.3). For details of resin microscopy and on-section immunocytochemistry, the reader is referred to Newman and Hobot (2001). The immunostaining quality of resin sections is usually comparable to that yielded by paraffin sections. Prior microwave heating of resin sections results in enhanced immunoreactivity with specific, easily interpretable staining using a variety of antibodies. In addition, resin sections allow immunogold and immunogold-silver immunostaining. Excess background staining is not a problem with resin sections, provided they are premicrowaved or heated by other means. In some cases, resin sections may show less intense staining than that exhibited by paraffin sections, which is due to thinness (~80 nm) of the former sections. Also, positive staining may not be achieved in some cases. This is exemplified by antibodies to neutrophil elastase and CD61, which show negative immunostaining on resin sections even after microwave heating (McCluggage et al., 1995). In contrast, immunostaining of CD20 is more reliable on resin sections than on paraffin sections of bone marrow trephine biopsy specimens. Note that the reaction of antibody with antigen is a surface phenomenon in resin sections. Various types of resins can be used for tissue embedding for antigen retrieval. Both water-miscible and water-immiscible resins (Hayat, 2000a) can be used in immunostaining for light and electron microscopy. Water-miscible resins used in light microscopy include the acrylic polymer glycol methacrylate (Suurmeijer and Boon, 1993b) and LR White (Sormunen and Leong, 1998), as well as the hydrophobic resins methyl methacrylate (Hand et al., 1996) and Polybed 812 (McCluggage et al., 1998). All these were used with prior microwave heating. Recently, using EDTA and heat, antigen retrieval was accomplished on Epon sections (Röcken and Roessner, 1999). The following embedding mixture is excellent when sections of thickness are required. It has been employed for embedding bone marrow trephine biopsy specimens for 155

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light microscopy (McCluggage et al., 1995): Polarbed 812 DDSA MNA DMP-30

50 g 32 g 21 g 2g

DDSA: dodecenylsuccinic anhydride MNA: methyl nadic anhydride DMP-30: 2, 4, 6, tris (dimethylaminomethyl) phenol

ROLE OF FIXATIVE AND EMBEDDING RESIN IN ANTIGEN RETRIEVAL It is well established that formaldehyde reacts with amino groups on protein side chains (Fig. 7.2), introducing mostly reversible protein crosslinks. Epoxy monomer reacts with the

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new hydroxyl groups introduced on the protein by formaldehyde; the epoxy molecules thereby are copolymerized with the protein. In other words, formaldehyde functions as a link between protein side groups and the epoxy monomer (Brorson et al., 1999). In contrast, such a copolymerization does not occur between acrylic resins and tissue proteins. These resins permeate the tissue without chemically binding to them. Accordingly, during thin sectioning, the two resins cleave differently. In the case of acrylic resins, the surface of cleavage tends to follow the path of least resistance; this path is the interface between the resin and proteins. Thus, more epitopes without splitting are exposed at the surface of acrylic sections. On the other hand, in the case of epoxy sections, the resistance in such interfaces is not significantly less than that in tissue proteins, which results in the splitting of protein

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molecules. Also, the surface of epoxy sections is smoother than that of acrylic sections. Consequently, fewer epitopes are exposed on the surface of the former sections. However, epoxy resins are of value, being easier to cut and more stable under the electron beam and better preserving the ultrastructure.

IMMUNOSTAINING OF THIN RESIN SECTIONS Antigen sites can be unmasked not only on thick and semithin resin sections for light microscopy but also on thin resin sections for electron microscopy. Antigen retrieval at the ultrastructural level has been accomplished on thin sections of epoxy resins (Stirling and Graff, 1995; Röcken and Roessner, 1999) and LR White resin (Wilson et al., 1996; Sormunen and Leong, 1998). Because epoxy and LR White resins are superior to some other resins with respect to preserving the cellular details and other characteristics, antigen retrieval methods using these two resins for electron microscopy are presented. For electron microscopy, tissues can be fixed with a mixture of formaldehyde and glutaraldehyde or with the latter only. Glutaraldehyde fixation better preserves cellular details but strongly masks antigens. However, antigenic sites can be unmasked on epoxy thin sections of glutaraldehyde-fixed tissues by exposing the sections to strong oxidizing agents such as EDTA, hydrogen peroxide, sodium methoxide, or sodium metaperiodate. These treatments also allow immunostaining of sections of postosmicated tissues by removing osmium bonds. Moreover, such treatments temporarily minimize the hydrophobicity of epoxy section surface and may increase resistance to heavy metal poststaining (Bendayan and Zollinger, 1983; Causton, 1985; Newman and Hobot, 1993). The above-mentioned etching pretreatments are generally useful for epoxy sections but not for acrylic (LR White) sections because unlike acrylic resins, epoxy resins form covalent bonds with proteins. In other words, epoxy resins copolymerize with the tissue, while acrylic resins surround the tissue components without becoming part of them. Accordingly, epoxy resins strongly mask the proteins that become mostly inaccessible to antibodies. Therefore, epoxy sections, especially of glutaraldehyde-fixed tissues, require etching to unmask the antigens. The surface of acrylic sections is rougher than that of epoxy sections. Moreover, acrylic sections are less crosslinked and more hydrophilic than epoxy sections. As a result, immunostaining reagents penetrate acrylic sections easily, facilitating antigen detection. Exposure of acrylic sections to oxidizing agents worsen both the known instability of these sections under the electron beam and the structural details. To facilitate the access of antigens to antibodies, the protocol of embedding and etching given on page 159 is used (Crowley, 1997). The sections of this low-crosslinked embedding medium are thought to allow easy penetration of aqueous immunostaining fluids. A saturated solution of sodium periodate is prepared by dissolving 1 g of this reagent in 5 ml of distilled water and passing the solution through a pore filter. The grids are wetted by floating them on drops of distilled water and then floated on drops of the sodium periodate solution for ~15 min (this duration can be changed to obtain maximum immunoreactivity). The grids are thoroughly rinsed in distilled water and must not be allowed to dry before immunostaining.

Antigen Retrieval on Resin Sections

Embedding Media: Araldite 502 Eponate 12 DDSA Dibutyl Phthalate DMP-30

159

15ml 25ml 55ml 1% 1.5%

If background staining is a problem and standard rinsing with PBS fails to reduce nonspecific staining, boosting the sodium chloride concentration from ~0.9% (150 mM) to ~4.5% (750 mM) may help (Chiovetti, 1998). After this treatment, the grids must be rinsed several times in standard PBS before further processing, so that the salt concentration is reduced to the range of physiological strength. As an example, the grid can be rinsed five times for ~2 min each on drops of high-salt buffer, followed by two rinses for ~2 min each on drops of standard salt buffer. This routine can be used after incubation in the primary antibody or any other incubation (e.g., secondary antibody incubation, colloidal gold) that is suspected of contributing to nonspecific background staining. Although the exact explanation for the beneficial effect of the high-salt concentration is not known, it may alter the conformation of protein molecules and change their overall charge, making them less likely to bind nonspecifically on the surface of the section. It is known that high salt concentrations tend to precipitate proteins out of the solution in biochemical studies and are also used to wash chromatography columns. Accordingly, only the antibody molecules that have been bound specifically to antigenic sites remain on the section surface in the presence of high salt concentrations. If cross reactivity is a problem during conjugated gold-antibody double labeling with monoclonal antibodies from the same animal (e.g., mouse monoclonals), it can be avoided by incubating very carefully first one side of the grid in one of the mouse monoclonals and then the other side of the grid in the second mouse monoclonal (Chiovetti, 1998). Precaution must be used to prevent sinking of the grid in drops of the incubation reagents. Hexagonal mesh, uncoated nickel grids should be used. To avoid the adverse effect of high temperatures on thin resin sections in the microwave oven, staining can be carried out at ~5°C in the microwave oven (HernándezChavarría and Vargas-Montero, 2001). Heat generated by microwave irradiation is dissipated by this approach. Rapid staining is accomplished by molecular vibrations in the microwave oven, which induce molecular collisions leading to accelerated chemical reactions. Thin resin sections of the tissue fixed with glutaraldehyde/osmium tetroxide are transferred onto a grid, which is then placed into a BEEM capsule. Six capsules are placed on a plastic support, which is placed into a 500-ml beaker containing ice cubes and 300 ml of tap water, covering the bottom of the capsules. It takes ~5 min to equilibrate the temperature in the ice bath to 5°C, which is maintained during staining in the microwave oven. The temperature is measured after each heating period, and ice cubes are added as melting occurs. The staining is carried out with of 4% uranyl acetate in 50% ethanol for 1 min in a microwave oven set at a power level of 125.6W, followed by rinsing with 500 ml of distilled water. This is followed by staining for 1 min with of triple lead citrate (Sato et al., 1988) and then rinsing with 500 ml of distilled water. This lead citrate staining solution avoids the production of artifactual lead carbonate precipitates.

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ANTIGEN RETRIEVAL ON SECTIONS OF MODIFIED EPOXY RESIN Because epoxy resins copolymerize with tissue proteins, and acrylic resins do not, sections of the former yield less immunostaining. However, to take advantage of the other superior characteristics of epoxy resins explained earlier, the immunostaining of sections of these resins can be enhanced by moderately increasing the proportion of the accelerator DMP-30 and microwave heating (Brorson, 1998a, b; Brorson et al., 1999). Conventional concentrations of accelerator in the epoxy mixture form abundant chemical bonds between resin and tissue. In contrast, a high concentration of accelerator reduces copolymerization of the epoxy resin with tissue proteins, while heating breaks down both protein crosslinkages introduced by aldehydes and the bonds between the resin and the tissue. The breakdown of abundant bonding with heating in the former case is insufficient to allow efficient access of the antibody to the antigen. Figure 7.3 shows the possible mechanism responsible for exposing epitopes to antibodies on the surface of thin epoxy sections after heating. Tissue specimens are fixed overnight at 4°C with a mixture of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). They are dehydrated in ethanol followed by propylene oxide. Infiltration is carried out in two steps using DMP-30 in concentrations of 4% and 2%, respectively, and embedding in the resin containing 2% DMP-30. The specimens in gelatin capsules are polymerized for 3 days at 56°C. Thin sections mounted on nickel grids are treated in 0.01 M citrate buffer (pH 6.0) for 15 min at 95°C in a PCR machine (GeneAmp 2400, Perkin Elmer). The sections are treated with 10% BSA in PBS (pH 7.2) for 4 hr to block nonspecific labeling. Incubation is carried out overnight at 4°C in the primary antibody, appropriately diluted in PBS. This is followed by washing three times for 5 min each in PBS and incubation for at 22°C in colloidal gold (15 nm)–conjugated secondary antibody, appropriately diluted in PBS containing 3% BSA. The sections are poststained with 5% uranyl

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acetate in 30% ethanol for 20 min and then with lead citrate for l0 min. The results of this procedure are shown in Figure 7.4.

EFFECT OF HEATING Heating is effective in antigen retrieval on semithin and thin sections of resin-embedded tissues. This results not only from the breakdown of protein crosslinks introduced by aldehyde but also from the breakage of bonds between the epoxy resin and the embedded tissue (see Fig. 7.2). It is known that epoxy resins form covalent bonds with tissue proteins during embedding. Microwave heating has been employed for antigen retrieval on thin sections of formaldehyde and tissues embedded in Araldite for electron microscopy (Stirling and Graff, 1995). In this study thin sections on grids were treated for 1 hr at room temperature in a humid chamber with a saturated aqueous solution of sodium metaperiodate to reverse the effects of The heat treatment was carried out on a hot plate. Treatment of thin sections with sodium ethoxide is not recommended, for it damages the ultrastructure. Microwave heating has also been used for antigen retrieval on thin sections of tissues fixed with glutaraldehyde and and embedded in LR White or TAAB resin (Wilson et al., 1996). In this study, compared with nonmicrowaved sections, microwave-treated thin sections revealed markedly enhanced gold labeling of type IV collagen in the oral epithelial basal lamina for both types of resins.

ANTIGEN RETRIEVAL ON THIN RESIN SECTIONS USING AUTOCLAVING In addition to antigen retrieval on sections of paraffin-embedded tissues for light microscopy, antigen retrieval can be carried out on thin resin sections for electron

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microscopy. Using heat pretreatment, antigen retrieval can be accomplished on paraffin sections and thin resin sections. The following method was used for immunostaining thin sections of tissue embedded in a resin (Xiao et al., 1996). Tissues are fixed with a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 6 hr at 20°C. They are embedded in Lowicryl K4M at –40°C. Thin sections are mounted on uncoated metal grids or synthetic grids and airdried. They are placed in 10 mM sodium citrate buffer (pH 6.0) and heated in an autoclave for l0 min at 120°C. After being cooked for 20–30 min at room temperature, the sections are rinsed in PBS (pH 7.4). The sections are immersed in PBS containing 0.1% BSA and 0.1% gelatin (PBSG) for 5 min, and then treated with 10% normal goat serum in PBSG for 10 min. This is followed by incubation in the primary antibody (appropriately diluted) in a humid chamber overnight at 4°C. The sections are rinsed in PBSG containing 0.1% Tween 20 and incubated for 1 hr in colloidal gold (15 nm)–labeled goat antimouse IgG diluted in 1:20 with PBSG containing

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0.1% Tween 20. Following rinsing in PBSG containing Tween 20, the sections are rinsed in PBSG and then in PBS. The sections are postfixed for l0 min with 2% glutaraldehyde in PBS and rinsed several times in PBS and distilled water; poststaining is carried out with uranyl acetate and lead citrate. For negative controls, the primary antibody is replaced with PBS. The results of this procedure are shown in Figure 7.5.

RAPID STAINING OF THIN RESIN SECTIONS IN MICROWAVE OVEN A microwave oven operating at 2,450 MHz with a maximum output power of 900 W and a cycle time of 2 sec can be used for rapid staining in a microwave oven (Cavusoglu et al., 1998). The oven contains a gas exhaust system and a built-in ceramic thermocouple temperature probe (PT 100). To determine the distribution of microwave heating, a piece of thermal paper is placed on the floor of the oven and subjected to microwave heating at 900 W for 1 min, and the hot spots are located. A glass bottle containing 40 ml of tap water is placed in the oven to measure the temperature during heating. The temperature of the water is monitored throughout the staining. Tissues are fixed with glutaraldehyde followed by and embedded in Epon. Thin sections are mounted onto a Formvar-coated grid, which is placed (section side down) on the surface of 4% aqueous solution of uranyl acetate in a staining dish. The dish is placed on the hot spot in the oven at a power of 600 W for 1 min at 20°C (initial temperature) to 94°C (final temperature). The dish is taken out of the oven and the grid is rinsed with distilled water. The grid is placed on the surface of lead citrate solution in the dish, which is placed in the oven and stained for 1 min at 20°C (initial temperature) to 93°C (final temperature). The results of this procedure are shown in Figure 7.6 (Cavusoglu et al., 1998). Figure 7.7 shows ultrarapid staining of biopsy heart tissue with uranyl acetate and lead citrate for 15 sec each in a microwave oven.

MICROWAVE HEAT-ASSISTED RAPID PROCESSING OF TISSUES FOR ELECTRON MICROSCOPY In certain diagnostic studies with the electron microscope, it is helpful to complete fixation and embedding as quickly as possible. This accelerated processing can be completed in ~2 hr. As shown in Figure 1.1B, the quality of cell preservation is satisfactory. The recommended protocols for routine processing, routine microwave processing, and vacuum microwave processing, respectively, are given in Table 7.1 (Giberson et al., 1997).

MICROWAVE HEAT–ASSISTED IMMUNOLABELING OF RESIN-EMBEDDED SECTIONS Conventional, high-resolution immunoelectron microscopy has been extensively used for the subcellular distribution of proteins to obtain information on their functions. However, this approach is time consuming. Processing time can be substantially reduced by applying microwave heating; the total time is reduced to 4–5 hr while the conventional

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method requires ~20 hr. All steps are carried out in a microwave oven. Tissues are fixed in a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol, and embedded in LR White resin for 75 min. Thin sections are incubated in primary antibody at 37°C for 15 min and then in colloidal gold–goat antirabbit IgG for 15 min at the same temperature (Rangell and Keller, 2000). Temperature should be strictly controlled in the microwave oven with a temperature probe that has a feedback mechanism to regulate the energy output of the microwave oven and thus maintains the optimal temperature. Alternatively, temperature can be controlled by placing a water load in the chamber of the microwave oven, which absorbs extra energy and provides humidity, slowing the evaporation of reagents. In addition, hot spots in the chamber should be avoided by using the neon bulb display method (Chapter 5). Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longer durations and higher temperatures may result in undesirable changes in antibody concentration and molarity of the salts and pH. After heat treatment, the sections should be kept at room temperature for at least 2 min to stabilize the antibody-antigen complexes. The step-by-step procedure for microwave heat-assisted immunolabeling of resin-embedded thin sections for electron microscopy follows (Rangell and Keller, 2000): 1. Fix the tissue in a mixture of 2% formaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 40 sec at 37°C in a microwave oven. 2. Rinse twice for 2 min each in the buffer at room temperature. 3. Dehydrate twice for 45 sec each at 45°C in a microwave oven. 4. Infiltrate for 15 min at 50°C with 1:1 mixture of 100% ethanol and LR white resin in a microwave oven. 5. Infiltrate three times for 10 min each at 50°C in a microwave oven. 6. Polymerize for 15 min at 95°C in a microwave oven using the temperature probe. 7. Polymerize for 1 hr at 95°C in a microwave oven without using the temperature probe. 8. Cut thin sections (may require 30 min). 9. Transfer sections onto grids and float on drops of PBS containing 0.1% Tween-20 (PBST) for 5 min at 37°C in a microwave oven. 10. Float the grids on drops of PBST containing 1 % bovine serum albumin and 0.1% coldwater fish skin gelatin (PBST+BG) for 5 min at 37°C in a microwave oven. 11. Incubate by floating sections three times for 5 min each at 37°C on drops of the primary antibody in PBST+BG in a microwave oven. 12. Keep the sections for 2 min at 37°C in a microwave oven to stabilize antibodyantigen complexes. 13. Rinse twice for 5 min each in PBST+BG at room temperature. 14. Incubate three times for 5 min each at 37°C on drops of the secondary antibodycolloidal gold complex in PBST+BG in a microwave oven. 15. Keep the sections for 2 min at 37°C in a microwave oven. 16. Rinse twice for 5 min each in PBST at room temperature. 17. Rinse twice for 5 min each in double-distilled water at room temperature. 18. Stain in 1% uranyl acetate for 30 sec at 37°C in a microwave oven. 19. Rinse twice for 5 min each in double-distilled water at room temperature. The total duration is ~4.3 hr.

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MICROWAVE HEAT-ASSISTED IMMUNOGOLD METHODS Both immunogold alone and silver-enhanced immunogold methods can be employed in combination with microwave heating for labeling antigens. The use of colloidal gold particles as markers for antigenicity has become truly universal (Hayat, 1989–1991, 2000a). The advantage of the immunogold–silver staining (IGSS) method over the immunogold technique is that the former allows the use of very small colloidal gold particles for detecting antigenicity with both light and electron microscopy (transmission and scanning electron microscopy) (Hayat, 1995). These methods are significantly more sensitive than standard immunoperoxidase procedures. The major benefit of combining immunogold methods with microwave heating is shortening the duration of incubation in the primary and secondary antibodies. The use of microwave heating also offers the potential for increasing the positive reaction product and decreasing the nonspecific background label. The immunogold method can also be employed in conjunction with EDTA and conventional heat for electron microscopy (Röcken and Roessner, 1999). In this study human autopsy tissue specimens were fixed with a mixture of 2% formaldehyde and 2.5% glutaraldehyde and embedded in Epon. Various etching and antigen retrieval techniques were tested. The ideal pretreatment for achieving increased immunogold staining of amyloid consisted of conventional heating of thin resin sections at 91 °C for 30 min in 1 mM EDTA (pH 8.0).

Immunogold-Silver Staining Jackson et al. (1988) were the first to employ the immunogold–silver staining (IGSS) method in combination with microwave heating. They completed within minutes the incubations in primary and secondary antibodies for detecting immunoglobulins in paraffin sections of human tonsil. van de Kant et al. (1990) applied the same method, except that resin instead of paraffin sections were used to detect bromodeoxyuridine incorporated in cells of the mouse testis. Tissue morphology is preserved better in a resin than in paraffin. The former also allows the use of thinner sections. Boon et al. (1989) used a similar procedure for staining beta-human chorionic gonadotropin in paraffin sections of the syncytiotrophoblast of first-trimester placenta. Recently, using the IGSS method, Taban and Cathieni (1995) visualized the goldprotein-ligand complex on cryostat sections of rat brain; this method can be used for light and electron microscopy.

Droplet Procedure Tissues are fixed with buffered formalin or Kryofix and embedded in paraffin (Boon et al., 1991). Sections ( thick) are transferred to a glass slide, deparaffinized, rehydrated, and washed in running tap water for l0 min. They are treated with Lugol’s iodine for 5 min and rinsed briefly in tap water. Following destaining with 2% aqueous sodium thiosulfate for 10–15 sec, the sections are washed in running tap water for l0 min.

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The sections are washed in two changes of 5 min each in Tris buffer I (2.5% NaCl and 0.55% Tween 20, diluted in 0.05 mol/liter Tris-HCl buffer, pH 8.2). The excess buffer is removed by wiping around the sections, which are then covered for l0 min with of normal goat serum (NGS) diluted 1:1 with PBS (pH 7.4). Excess NGF is removed by wiping around the sections. The sections are covered with of primary antibody appropriately diluted in PBS (pH 7.4) containing 0.05% BSA (freshly prepared). The slide is placed on the polystyrene platform in the microwave oven and heated at 50% power for 5 min. A water load of 200 ml tap water has already been placed in the oven. The sections are washed with Tris buffer for l0 min, followed by washing in two changes of l0 min each in Tris buffer II (0.05 mol/liter Tris-HCl buffer, pH 8.2). Excess buffer is removed by wiping around the sections, which are then covered with of NGS for 10 min at room temperature. Excess NGS is removed, and the sections are covered with of colloidal gold conjugated secondary antibody. The slide is placed on the polystyrene platform in the microwave oven and heated at 50% for 5 min; the oven contains a water load of 200 ml of tap water. The sections are washed in three changes of 5 min each in Tris buffer II, rinsed with distilled water, and washed three times of 3 min each in distilled water. After excess water is removed, the sections are covered with of silver enhancement mixture for 8–11 min at 20°C. The silver enhancement mixture is prepared immediately before use by mixing equal volumes of the enhancer and initiator solutions of the Janssen Intense™ LM kit. The sections are washed three times for 5 min in distilled water, dehydrated, and mounted.

Chapter 8

General Methods of Antigen Retrieval

GENERAL PROCEDURE FOR ANTIGEN RETRIEVAL USING MICROWAVE HEATING (See Fig. 8.1.) 1. Fix the tissue without delay for 4–6 hr in 4% buffered formalin; the longer the tissue remains in the fixative, the lesser the chances of epitope retrieval (Hayat, 2000a,b). 2. Wash in several changes of PBS; if available, an Autotecnicon should be used in this step and in steps 3 and 4 below. 3. Dehydrate in a series of ascending concentrations of ethanol. 4. Infiltrate and embed in paraffin. sections with a microtome and float them on a water bath kept at 5. Cut room temperature so as to stretch the sections. An ordinary glass slide is used to transfer the sections onto another water bath kept at 58°C to further stretch the sections. Lift them by the SuperFrost slides, thus mounting them in the process. The sections are allowed to dry in an upright position in a slide holder at a temperature of