Fine Structure Immunocytochemistry

476 Pages·1993·14.922 MB·English

by  Dr. Gareth Griffiths (auth.)

(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
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© 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
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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.