MICROWAVETECHNIQUESAND PROTOCOLS
MICROWAVETECHNIQUES AND PROTOCOLS Edited by
RICHARDT. GIBERSON, MS Ted Pella Inc., Redding, CA
and
RICHARD S. DEMAREEJR., PhD California State University, Chico, CA
HUMANA PRESS TOTOWA,NEWJERSEY
© 2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by: Patricia Cleary. Cover illustration: From Fig. 3A in Chapter 12 "In Vivo Microwave-Assisted Labeling of Allium and Drosophila Nuclei" by Mark A. Sanders and David M. Gartner. Production Editor: Kim Hoather-Potter. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail:
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Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is [0-89603-903-X/01 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Microwave techniques and protocols / edited by Richard T. Giberson and Richard S. Demaree, Jr. p. cm. Includes bibliographical references and index. ISBN 0-89603-903-X (alk. paper) 1. Microscopy--Technique. 2. Microwaves. 3. Electron microscopy. 4. Microwave drying. 5. Microwave devices. I. Giberson, Richard T. II. Demaree, Richard S. QH207.M59 2001 570'. 28'25--dc21
2001016992
PREFACE Microwave Techniques and Protocols brings to the clinical and research community a how-to manual based on contributions from laboratories that are using microwaves as a means to facilitate biological sample processing. This project actually began in 1992 when Ted Pella Inc. decided to develop microwave technology for clinical and research applications. A number of questions arose almost immediately: What can microwave technology do to improve sample processing for microscopy applications? How does it work? And, most importantly, why use it? In 1992 there was adequate literature to engender confidence that microwaves could be used to facilitate the following: (1) microwaveassisted stabilization or chemical fixation for light and electron microscopy, (2) enhanced special stain protocols for histology, (3) accelerated decalcification, (4) lower incubation times during immunocytochemistry, and (5) reduced processing times for small tissue biopsies into paraffin. The problem was where to begin when you didn't know anyone in the field. Microwave-assisted chemical fixation presented itself as the place to start to the literature interest at that time and owing to the fact that success or failure could be determined relatively quickly. It took almost three years before publication of our first paper on microwave fixation for electron microscopy (Giberson and Demaree, 1995). By 1995 we still could not locate anyone using the microwave to routinely fix tissue, and based on our experience, we could understand why. Success had come with a lot of effort, but few fundamental answers as to how or why the process worked. Much has changed since that original paper. Protocols now exist for microwave-assisted chemical fixation for both light and electron microscopy that can be done rapidly, reproducibly, and routinely. However, fixation is only a fraction of the time required in overall sample processing. The original 1995 paper was the seed for moving forward and demonstrating that the microwave could be used for each step in processing for electron microscopy (Giberson et al., 1997). That 1997 paper described a four-hour protocol that has since been shortened to two hours (see Chapter 2). A microwave workshop series that began in the summer of 1995, and continues to this day, is the basis for the contributions to this book.
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Preface
The contributors to this manual are uniformly from those laboratories routinely using microwave technology to facilitate their processing methods in the various fields of microscopy. The methods and results these authors describe are the tangible evidence that microwaves can be used routinely as the basis for improved sample processing for microscopy applications. These applications include complete sample processing protocols for light and electron microscopy, decalcification, and immunocytochemistry. The overall time savings, ease of use, and quality of results serve as justification for using microwaves in the laboratory. The question as to whether there is a "microwave effect" is alluded to, but not discussed in any great detail. When the term microwave technology is used, it is generic and intended to mean equipment designed for laboratory versus household use. Microwave Techniques and Protocols is designed for anyone with a background and experience in sample processing for immunocytochemistry, decalcification, light microscopy, or electron microscopy, and clearly demonstrates that microwave technology has a place in today's laboratory. Richard T. Giberson, Ms Richard S. Demaree Jr., VhD
REFERENCES Giberson, R.T. and Demaree, R.S., Jr. (1995) Microwave fixation: understanding the variables to achieve rapid reproducible results. Microsc Res Tech 32:246-254. Giberson, R.T., Demaree, R.S., Jr., and Nordhausen, R.W. (1997) Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique. J Vet Diagn Invest 9:61-67.
CONTENTS Preface ..................................................................................................
v
Contributors ........................................................................................
ix
Overview of Microwave-Assisted Tissue Processing for Transmission Electron Microscopy R i c h a r d S. Demaree, Jr. a n d Richard T. Giberson ....................................... 1
Vacuum-Assisted Microwave Processing of Animal Tissues for Electron Microscopy Richard T. Giberson ................................................. 13
Vacuum-Microwave Combination for Processing Plant Tissues for Electron Microscopy William A. Russin a n d Christina L. Trivett ............ 25
Basic Procedure for Electron Microscopy Processing and Staining in Clinical Laboratory Using Microwave Oven R o n a l d L. Austin ....................................................... 37
Specimen Preparation for Thin-Section Electron Microscopy Utilizing Microwave-Assisted Rapid Processing in a Veterinary Diagnostic Laboratory Robert W. N o r d h a u s e n a n d Bradd C. Barr ............ 49
6
Microwave Processing of Archived Pathology Specimens for Ultrastructural Examination Robert J. M u n n a n d Phillip J. Vogt ........................ 67
7
Microwave Fixation of Rat Hippocampal Slices Marcia D. Feinberg, Karen M. Szumowski, and Kristen M. Harris ......................................... 75
Microwave Processing Techniques for Biological Samples in a Service Laboratory Lou A n n Miller ......................................................... 89
°° VII
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Contents Microwave-Accelerated Decalcification: Useful Methods for Research and Clinical Laboratories Victoria J. Madden ................................................. 101 10
Microwave Processing of Sediment Samples Dawn Lavoie, Janet Watkins, and Yoko Furukawa .......................................... 123
11
Microwave Polymerization in Thin Layers of London Resin White Allows Selection of Specimens for Immunogold Labeling Jennifer E. Lonsdale, Kent L. McDonald, and Russell L. Jones ......................................... 139
12
In Vivo Microwave-Assisted Labeling of Allium and Drosophila Nuclei Mark A. Sanders and David M. Gartner ............... 155
13
Microwave-Assisted Cytochemistry: Accelerated Visualization of Acetylcholinesterase at Motor Endplates John P. Petrali and Kenneth R. Mills ................... 165
14
Microwave-Assisted Immunoelectron Microscopy of Skin: Localization of Laminin, Type IV Collagen, and Bullous Pemphigoid Antigen John P. Petrali and Kenneth R. Mills ................... 173
15
Microwave Paraffin Techniques for Botanical Tissues Denise Schichnes, Jeffrey A. Nemson, and Steven E. Ruzin .......................................... 181
16
Microwave-Assisted Formalin Fixation of Fresh Tissue: A Comparative Study Richard T. Giberson and Douglas E. Elliott ........ 191
17
Microwave-Assisted Processing of Biological Samples for Scanning Electron Microscopy Richard S. Demaree, Jr. ......................................... 209
Index ................................................................................................
217
CONTRIBUTORS RONALD L. AUSTIN " L S U Medical Center, Shreveport, BRADD C. BARR ° California Animal Health and Food
LA Safety Laboratory, School of Veterinary Medicine, University of California, Davis, CA RICHARD S. DEMAREE, JR. " D e p a r t m e n t of Biological Sciences, California State University, Chico, CA DOUGLAS E. ELLIOTT ° Ted Pella Inc., Redding, CA MARCIA D. FEINBERG " Department of Biology, Boston University, Boston, MA YOKO FURUKAWA • Naval Research Laboratory, Stennis Space Center, MS DAVID M. GARTNER " University of Minnesota, St. Paul, MN RICHARD T. GIBERSON • Ted Pella Inc., Redding, CA KRISTEN M. HARRIS ° Department of Biology, Boston University, Boston, MA RUSSELL L. JONES ° University of California, Berkeley, CA DAWN LAVOIE ° Naval Research Laboratory, Stennis Space Center, MS J E ~ - ~ R E. LONSDALE ° Department of Biology, University of California, San Diego, CA VICTORIA J. MADDEN • Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC KENT L. McDONALD ° Department of Biology, Electron Microscopy Laboratory, University of California, Berkeley, CA Lou ANy MILLER ° Centerfor Microscopic Imaging, College of Veterinary Medicine, University of Illinois, Urbana-Champaign, IL KENNETH R. MILLS • US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD ROBERT J. MUNN ° Department of Medical Pathology, University of California, Davis, CA JEFFREY A. NEMSON ° Department of Plant and Microbial Biology, University of California, Berkeley, CA ROBERT W. NORDHAUSEN ° California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California, Davis, CA ix
x
Contributors
Comparative Medicine Division, Aberdeen Proving
JOHN P. PETRALI *
Ground, MD Department of Plant Pathology, University of Wisconsin, Madison, WI STEVEN E. RUZIN Biological Imaging Facility, College of Natural Resources, University of California, Berkeley, CA MARK A. SANDERS • Imaging Center, College of Biological Sciences, University of Minnesota, St. Paul, MN DENISE SCHICHNES • Biological Imaging Facility, College of Natural Resources, University of California, Berkeley, CA KAREN M. SZUMOWSKI • Department of Biology, Boston University, Boston, MA CHRISTINA L. TRIVETT Department of Botany, Weber State University, Ogden, UT PHILLIP J. VOGT * Department of Medical Pathology, University of California, Davis, CA JANET WATKINS GB Tech Inc., Naval Research Laboratory, Stennis Space Center, MS
WILLIAM A. RUSSIN ° •
•
"
1
Overview of Microwave-Assisted Tissue Processing for Transmission Electron Microscopy Richard S. Demaree, Jr. and Richard T. Giberson
INTRODUCTION The purpose of this book is to provide new, reliable, and recently updated protocols for processing many different kinds of samples, using microwave (MW) technology. Chapters included deal with a wide variety of methods and samples, everything from paraffin processing for light microscopy (LM) to scanning and transmission electron microscopy (TEM), to immunocytochemistry. MW-assisted processing of microscopy samples has a long, convoluted history beginning with the pioneering efforts of Mayers (1970) and Zimmerman and Raney (1972). The first LM and TEM report, utilizing MW-enhanced aldehyde fixation of tissues, was by Login and Dvorak (1982). Of the scattered reports that then began appearing in the literature, most reported using MW-assisted processing for the fixation step. Login et al. have chronicled these events in a series of reviews (Login and Dvorak, 1993; 1994; Login et al., 1996). In 1995, Giberson and Demaree reported ice-encased fixation in the MW as a means to control the effects of heating for TEM sample processing. Shortly thereafter, the authors described protocols for rapid, reliable MW polymerization of resin (Demaree et al., 1995) and complete MW tissue processing from fixation to polymerized resin blocks (Giberson and Demaree, 1995). In 1997, an updated, complete From: Microwave Techniques and Protocols Edited by: R. T. Giberson and R. S. Demaree Jr. © Humana Press Inc., Totowa, NJ
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Demaree and Giberson
protocol was reported, utilizing microcentrifuge tubes (MT), which no longer required ice for cooling samples (Giberson et al:, 1997). Since 1997, the variety of MW protocols and samples processed has expanded rapidly, e.g., scanning electron microscopy (Fox and Demaree, 1999). Because much of this research is either unpublished or appears in a wide variety of journals, the authors felt that it was time to present a compendium of state-of-the-art MW sample processing. For tissues that require routine processing, Giberson et al. (1997) and Giberson and Demaree (1999) publications are a good starting point. Special adaptations for more difficult tissues are covered in this and subsequent chapters. Protocols for LM (Chapters 12, 15, and 16), scanning electron microscopy (Chapter 17), immunocytochemistry (Chapters 12-15), and decalcification (Chapter 9) are presented. Specific applications, such as clinical pathology (Chapters 4-6) are also covered. The remainder of this chapter presents updated suggestions and typical results from MW-processed tissues for TEM. MATERIALS AND M E T H O D S
Protocol Outline for TEM The steps in the protocol and the times required for each are shown below. The designation "MW" preceding a step, indicates that it is performed in the MW oven. This is an updated protocol based on earlier work (Giberson and Demaree, 1995; Giberson et al., 1997; Giberson and Demaree, 1999). Micrographs presented in the Results came from original work by the authors, or from contributed work of other MW users. A Model 3450 Microwave from Ted Pella was used for all MW processing steps. All the MW accessories used during the various processing steps are available from Ted Pella. During the fixation steps samples are processed in 1.7 mL MTs containing 600 ktL liquid. A Teflon ® holder for the MTs is placed in the established cold spot for MW processing. In step 7, specimens are transferred from the MTs to solvent resistent flowthrough baskets that are placed in 60 × 15 mm polypropylene Petri dishes containing approx 15 mL reagent (Giberson et al., 1997). MW-assisted resin polymerization is done underwater, in BEEM T M capsules sealed with Parafilm ®M, and held in place using a specially designed Teflon rack.
Determination of Sample Location Within MW The creation of a large cold spot, a region of homogenous MW heating, evolved from earlier work (Giberson and Demaree, 1995; Giberson
Overview of MW Processing for TEM et al., 1997). Two water loads (800-mL plastic beakers) are positioned in the MW cavity, so that the neon bulb array, which is 3 in. wide, will fit between the two beakers. The water load on the right is recirculated and cooled by the load cooler. Water-load placement and volume are adjusted until all, or nearly all, of the lights in the array do not light during 10 s MW exposure. These locations are marked on a grid map taped to the oven floor. The uncirculated water load is changed when it becomes hot to the touch (--50°C).
M W Processing Steps for TEM 1. MW aldehyde fixation (tissue in fix > 1 h: may be done under vacuum [vac]: see Chapters 2 and 3). Time: --2 rain. a. Fixative cooled to between 10 and 15°C. b. MW for 10-20-10 (10 s at 100% power; 20 s at 0% power; 10 s at 100% power) (temperature [temp] control is not used during fixation; place the temp probe in the water-load beaker). Note: In small fluid volumes, the temp probe can act as a MW antenna, which will cause excessive heating, compared to the other tubes. This will cause poor and/or unequal fixation between samples. 2. MW aldehyde fixation (tissue in fix
