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Fine Structure Immunocytochemistry PDF

476 Pages·1993·14.922 MB·English
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(Jareth (Jriffiths Fine Structure Immunocytochemistry With Contributions by Brian Burke and John Lucocq With 90 Figures Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest Dr. GARETH GRIFFITHS European Molecular Biology Laboratory Postfach 102209 W-6900 Heidelberg, Germany Front Cover: A thawed cryo-section (Tokuyasu technique) of a rabbit kidney cell (RK-13) infected with vaccinia virus. The section was labelled with an antibody against P65, the target protein of the drug rifampicin which blocks virus assembly. This protein is localized to the spherical immature virus but only poorly to the mature, brick-shaped particles. From a study by B. Sodeik, M. Ericsson, B. Doms, B. Moss, and G. Griffiths. ISBN-13: 978-3-642-77097-5 e-ISBN-13: 978-3-642-77095-1 DOl: 10.1007/978-3-642-77095-1 Library of Congress Cataloging-in-Publication Data. Griffiths, Gareth. Fine structure immunocyto chemistry / Gareth Griffiths with contributions by Brian Burke and John Lucocq. p. cm. Includes bibliographical references and index. ISBN 3-540-54805-X (Berlin) ISBN 0-387-54805-X (New York) 1. Immunocytochemistry. 2. Electron microscopic immunocyto chemistry. 1. Burke, Brian. II. Lucocq, John. III. Title. QR187.I45G75 1992 574.8T6042--dc20 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1993 Softcover reprint of the hardcover I st edition 1993 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Media conversion: Elsner & Behrens GmbH, Oftersheim 31/3145-5432 I 0 - Printed on acid-free paper To Tok and Jan for their teaching, inspiration and friendship Preface Electron microscopy in the biological sciences can be divided into two disciplines. The first, concerned with high resolution detail of particles or periodic structures, is mostly based on sound theoretical principles of physics. The second, by far the larger discipline, is interested in the information obtainable from thin sections. The theoretical back ground to those groups of techniques for preparing and looking at thin sections is often inexact and "loose", for want of a better word. What should be chemistry is often closer to alchemy. This kind of electron microscopy is often enshrined with mystical recipes, handed down from generation to generation. Admittedly, many of the processes involved, such as those required to embed tissue in epoxy resins, involve multiple interconnected steps, which make it difficult to follow the details of anyone of these steps. If all these steps are shrouded in some mystery, however, can one really trust the final image that emerges on the EM screen? When we present the data in some semi quantitative form is there really no better way to do it than to categorize the parameters with ++, +/-, etc? What happens when one labels the sections with antibodies? Does the whole business necess arily need to be more of an "art" than a "science"? Upon reflecting on these problems in 1981, I had the impression that many of the multi-authored textbooks that existed then (and that have appeared since) tended to exacerbate or at least perpetuate this problem. They are too often "heavy" on recipes and "light" on theoretical background. To be fair, the latter sometimes hardly exists. In many cases, however, I became convinced that there was more theoretical information available than was making its way into the main textbooks. In 1981, against this background, I decided to write my own book. Having made that decision I found there was no turning back, for the next 10 years. It was during this prolonged effort that I was forced to realize why no such book already existed! I must admit that I have changed my mind about a number of things since I started this project. The most important concession concerns the use of the new generation plastic resins, especially the Lowicryl resins. For groups such as ours, interested predominantly in the molecular cell biology of membrane proteins, the Tokuyasu cryosection method was the logical first choice for immunolabelling. VIII Preface However, it is becoming increasingly clear that for many problems these new resins offer a number of advantages over the cryosection method, especially in combination with freeze-substitution. Accord ingly, the book aims at giving a balanced view with respect to these two major approaches. The important theoretical and practical aspects relevant to these techniques are covered in detail; namely freezing, chemical fixation, antibodies, particulate markers and labelling reactions, both with antibodies and with the main, non-immunological approaches, such as the avidin-biotin system. During the 10 years it took to complete this book, I found the time available for this project decreasing every year, as my responsibilities as part of an active research team in the EMBL Cell Biology Program increased. A few years ago I realized I might never finish the book without some serious help. For this I am extremely grateful to two good friends for stepping in and writing two of the chapters. For the antibody chapter, the choice of Brian Burke was a logical one since Brian, along with Daniel Louvard, had been instrumental in setting up antibody facilities at EMBL. He was also especially aware of the problems involved in immunocytochemistry. For the chapter on particulate markers, John Lucocq was also an obvious candidate since he had published a number of innovative papers on different aspects of the use of colloidal gold. Three of the chapters I wrote were especially difficult for me. The first, on fixation, is a monster. When one starts to read the vast literature on this subject it is akin to jumping into a bottomless pit. The number of papers is almost infinite. In every citation one follows to a remote journal one finds more interesting references in ever more remote journals. Many of these papers are difficult to ignore. The problem was to see the "forest for the trees", to try to pull out concepts, rules or even guidelines from these hundreds of papers. I am not sure I have succeeded in this and if I did not, it was surely not due to lack of effort. I believe that the problem of chemical fixation is by far the most difficult we face in all of electron microscopy. The second chapter that gave me difficulties was Chapter 7 on immunolabelling. The idea I had was straightforward and obvious - to give a theoretical background to labelling reactions on sections. Although there is an enormous amount of literature on immunocyto chemistry, I found it extremely difficult to be able to select out the basic concepts, rules or guidelines. It appears that most of the literature involves practical rather than theoretical descriptions of labelling reactions. I found the existing textbooks far from satisfac tory. The appearance of the book by Larrson in 1988 was extremely satisfying and gave me a number of ideas. Before this time, however, I found much of the relevant information in the classical period of immunocytochemistry, especially in the papers by Coons' group in the Preface IX 1950s. With respect to theoretical principles, I would even go as far as to say that since the 1950s, as the number of papers in the field has increased, the quality of the average paper has deteriorated. Perhaps, diluted is a better word. Many of the relevant facts are buried in the Materials and Methods sections of a multitude of papers covering all the biological and medical sciences. In this respect it seems a fair guess that I will have missed a number of important references. For this I apologize and hope that I can rectify this state of affairs in future. Last, but not least of my problem chapters was the chapter on quantitation which is in two parts - stereology and quantitation of immunolabelling. Both parts had their own problems. For the stereology I must give the background for my rationale. From 1980 I was able to learn and apply basic stereo logical methods with the help of Hans Hoppeler in Professor Ewald Weibel's laboratory in Bern. The more I realized how powerful the stereological approach could be, the more surprised I became at how little these techniques are used, in particular for many problems which are crying out for their use. This is especially true in molecular cell biology, where this approach is often considered a mysterious one for getting a few esoteric estimates of surface or volume estimates that are not used in an integrated cell biological context. This state of affairs is even more frustrating when one becomes a ware of the fact that, during the past decade, the field of stereology has witnessed a true revolution in new concepts. When applied, these concepts can open up totally new directions in many research problems. I have often wondered why so many biologists in EM are so ignorant of even the basics of stereology. My only suggestion is that it stems, in part, from the fear one feels when one simply glances at an average theoretical stereol ogy paper. Stereo logy is a very precise, mathematically based discipline, and this is necessarily reflected in the language in which the key theoretical papers are written. There seems little doubt that the strictness of logic, the need to think in three dimensions and the presence of obscure-looking mathematical formu lae tend to frighten away many biologists. It is a feeling I have had myself, especially in the early days and it often returns when I am faced with a new concept such as the use of "vertical sections" or the "disector". In my own case, being able to put simple questions to the stereologists usually bridges the barrier between fog and clarity. When one gets the message, that message is almost invariably simple, and very elegant. Then, upon going back and re-reading the original paper the language becomes clear, despite all the fearsome formulae. In Chapter 11 I have attempted to both focus on the stereological aspects that are most relevant for molecular cell biology and to try to simplify the language. This is a risk, because by simplifying there is always the danger of over-simplifying and muddling up the well thought-out stereological rules. For this I was careful enough to work X Preface closely with top stereologists (see Acknowledgements). My main aim with the stereology part of Chapter 11 was to enable the reader to overcome his/her fear of this group of extremely powerful methods. If I succeed, that reader will then go to the original literature, will consult a stereologist and, ideally, will take one of the many stereology courses that are offered around the world. Finally, I should emphasize that since I attempted to dig into the theoretical concepts behind the various techniques discussed in this book I could not avoid being very critical of the immunoperoxidase method. I feel that this approach, more than any, exemplifies the problem of alchemy and loose thinking in our field. How often have I heard serious discussions about the idea of punching precise holes in membranes for antibodies to penetrate without really affecting the fine structure of cells! Nevertheless, since this approach is still widely used, I have attempted to give some general guidelines to this procedure, as well as the more straightforward "cell ghost" methods, and to delve into the theory behind the use of detergents and other reagents for permeabilization. At all stages of this book I have tried to consult the best specialists in each field. On this score I believe I have succeeded and to all these people lowe an enormous debt. GARETH GRIFFITHS Acknowledgements My extreme gratitude to all the following people, listed in no particular order: To all my friends and colleagues in the Cell Biology Program in EMBL for many things, especially ideas. A special thanks to our Cell Biology Program Coordinator, Kai Simons, and our DireCtor General, Lennart Philipson, for providing the atmosphere which has been so important for me. I must also thank Kai for his help with the detergents in Chapter 10. To Tim Johnson, whom I first met in 1983 and who changed my whole way of thinking about aldehyde fixation. Tim also helped me on a number of drafts of Chapter 3 and contributed enormously to the chapter. Thanks are also due to Werner Baschong for educating me about formaldehyde and for all his help with Chapter 3. To Brian Burke and John Lucocq for writing Chapters 6 and 8 respectively. Brian also critically read Chapter 7 while John raised important points on Chapter 11 as well as Chapters 4, 5 and 7. To Kiyoteru Tokuyasu and Jan Slot, to whom this book is dedicated. From both these good friends I have learned an enormous amount relating to many different aspects of this book. In fact, I am still learning from them each time we meet. To Jaques Dubochet for giving me the benefit of his wisdom on all aspects of freezing and for pointing out how "water is beautiful". Jaques also helped a lot with Chapter 5. To Rob Parton for critically reading the whole book, and for his many suggestions in the past 3 years. To Horst Robenek for all his help with the cryo and replica methods for immunolabelling. To John Gilkey, Eduard Kellenberger, Werner Villiger and Wim Voorhout for all their suggestions and information concerning Chapter 4. To Heinz Schwarz and York-Dieter Stierhof a separate thanks for educating me on the comparison between the different specimen preparation methods with respect to immunolabelling and for critically reading many chapters. XII Acknowledgements To David Vaux for his help with Chapters 6 and 7 and for writing the section on the use of recombinant DNA technology in Chapter 6. To Lars-Inge Larrson for writing his book and for all his suggestions in a number of chapters, especially Chapter 7. To Hubert Reggio and J an De Mey for their suggestions and criticisms of a number of chapters, especially Chapter 10. To Stuart Kornfeld and Mathias Uhlen for their expertise and ideas concerning lectins and protein A, respectively. A special word of thanks to my stereology friends. First, to Hans Hoppeler, Ewald Weibel and Luis Cruz-Orive for teaching me the basics of stereo logy. Hans was kind enough to read the first four or five drafts of Chapter 11. In retrospect, those early drafts were pisasters, and I am grateful to Hans for being so tolerant and understanding. To Torsten Mattfeldt, Terry Mayhew and Luis Cruz-Orive, who, between them, had many suggestions and constructive criticisms over many drafts of this chapter. Finally, to Hans-Jiirgen Gundersen, a special thanks for all his time, ideas, his surgical intervention and for not worrying about my feelings. To Kent Christensen, Paul Webster and Norbert Roos for a lot of help and ideas with many chapters. Kent and Paul together helped to write the historical background to the cryosection technique. Paul was also kind enough to allow me to put his unpublished glass-knife method in Chapter 5. To Edith Elliott and Clive Dennison for proof-reading the whole book. To Edith I am grateful for all her constructive comments and especially for her suggestions for a major reorganization of Chapter 5. To Francis Barr, Neil Emans and Beate Sodeik for proof-reading the whole book and for making many useful suggestions. To those specialists who read and corrected selected chapters; Allan Mackenzie (Chapters 2 and 5), John Tooze (Chap. 3), Alasdair McDowall (Chap. 5), Michael Smith (Chap. 5), Herb Hagler (Chap. 5) and Marc Horrisberger (Chap. 8). To all those who kindly contributed micrographs for the book. They are all individually acknowledged. To the support staff of EMBL. Foremost, to the many secretaries who typed the multiple drafts of each chapter, especially Julia Pickles, Rachel Wainwright and Anne Walter. This was truly an enormous effort. To our librarians Mary Holmes and Sue Mottram, who found those hundreds of references for me. To our drawing office, especially Petra Riedinger and Sigrid Bednarczyk, for all their help.

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Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.