Histological and histochemical methods : theory and practice

588 Pages·2015·6.886 MB·English

by  Kiernan, John Alan

5th Edition
Histological and
Histochemical
Methods
Theory and
practice
J. A. Kiernan
5th Edition
Histological and
Histochemical
Methods
RELATED TITLES
FROM SCION PUBLISHING
5th Edition
Histological and
Histochemical
Methods
Theory and Practice
J. A. Kiernan
Department of Anatomy and Cell Biology,
The University of Western Ontario, London, Ontario, Canada
Fifth edition © Scion Publishing Ltd, 2015
ISBN 978 1 907904 32 5
Fourth edition published 2008 (Scion Publishing)
Third edition published 1999 (Butterworth Heinemann)
Second edition published 1990 (Pergamon Press)
First edition published 1981 (Pergamon Press)
Scion Publishing Limited
The Old Hayloft, Vantage Business Park, Bloxham Road, Banbury OX16 9UX, UK
www.scionpublishing.com
Important Note from the Publisher
The information contained within this book was obtained by Scion Publishing Ltd from sources
believed by us to be reliable. However, while every effort has been made to ensure its accuracy,
no responsibility for loss or injury whatsoever occasioned to any person acting or refraining from
action as a result of information contained herein can be accepted by the authors or publishers.
Readers are reminded that medicine is a constantly evolving science and while the authors
and publishers have ensured that all dosages, applications and practices are based on current
indications, there may be specific practices which differ between communities. You should always
follow the guidelines laid down by the manufacturers of specific products and the relevant
authorities in the country in which you are practising.
Although every effort has been made to ensure that all owners of copyright material have been
acknowledged in this publication, we would be pleased to acknowledge in subsequent reprints
or editions any omissions brought to our attention.

Registered names, trademarks, etc. used in this book, even when not marked as such, are not to
be considered unprotected by law.
Typeset by Phoenix Photosetting, Chatham, Kent, UK
Printed in the UK
Cover images:
Top – cerebellar cortex of a rat, stained with the Holmes silver method for axons (Chapter 18) and
counterstained with neutral red at pH 4 (Chapter 6). Axons are black; nuclei are red.
Middle – trachea of a rodent, stained by Heidenhain’s AZAN trichrome method (Chapter 8). Nuclei
are red; cytoplasm is pink; cilia of the epithelial cells are orange; collagen and cartilage matrix are
various shades of blue. (Fixation by vascular perfusion has removed erythrocytes, which would
have stained orange.)
Bottom – vascular bundle in a dock (Rumex sp.) stained with safranine O and fast green FCF
(Chapter 6). Lignified cell walls of the xylem are red; cellulose cell walls of the surrounding
parenchyma are green.
Contents
Preface to the fifth edition vii
Acknowledgements ix
Conventions and abbreviations xi
1. Introduction to microtechnique 1
2. Fixation 12
3. Decalcification and other treatments for hard tissues 45
4. Processing and mounting 52
5. Dyes 72
6. Staining with dyes in one or two colours 137
7. Staining blood and other cell suspensions 170
8. Methods for connective tissue 184
9. Methods for nucleic acids 206
10. Organic functional groups and protein histochemistry 231
11. Carbohydrate histochemistry 263
12. Lipids 293
13. Methods for pigments and inorganic ions 321
14. Enzyme histochemistry: general considerations 342
15. Hydrolytic enzymes 348
16. Oxidoreductases 371
17. M ethods for soluble organic compounds of low molecular weight 406
18. Metal reduction and precipitation methods 419
19 Immunohistochemistry 454
20. Miscellaneous data 491
Bibliography 505
Glossary 549
Index 555

Preface to the fifth edition
How to use this book
Even if you are an experienced research worker or technologist, please READ CHAPTER 1,
ESPECIALLY Section 1.6, BEFORE ATTEMPTING TO CARRY OUT ANY PRACTICAL
INSTRUCTIONS, and look at the list of Conventions and abbreviations just before Chapter 1.
Otherwise, go directly to any subject that interests you, by way of the Contents list or the Index.
There are many cross-references to numbered sections of other chapters. Nobody reads this kind
of book from beginning to end.
The purpose of this book is to teach the chemical, physical and biological principles of fixation,
staining and histochemistry. I urge the reader always to determine the reason for every step in
a method before doing it. The theoretical explanations and practical instructions are therefore
closely integrated. This is to encourage an intelligent approach to microtechnique, in which the
user reviews the rationale of each new method instead of following a list of poorly understood
instructions. There is a reason for each instruction, and the printed procedural details may not
apply equally to all specimens. Adaptations and adjustments are often necessary, and are likely to
be successful only when they are justified by knowledge and understanding.
The reader requires some knowledge of chemistry (descriptive rather than mathematical) and
biology (cells, tissues, bacteria, fungi, etc.) to use this book effectively.
Readers, especially graduate students and others involved in research, are urged to follow up
references relating to the methods they are using. Do not go straight to the technical instructions
without reading about how a method is supposed to work. A textbook cannot provide all the
information, and there is often controversy about the best way to prepare specimens for particular
tests. Preparative procedures include collection of specimens (delays, drying, etc.), fixation
(type, time, temperature) and processing into an embedding medium (solvents, media, times,
temperatures, etc). Should sections of an unfixed specimen be frozen or somehow fixed before
staining with a dye or carrying out enzyme activity histochemistry? Before staining to make a
clinical diagnosis, should a film or smear of cells on a glass slide be air-dried or chemically fixed,
and if the latter, how? It is currently fashionable to call these combinations of chemicals and
conditions ‘preanalytical variables’, and it is recognized that they greatly influence such commonly
used procedures as staining with mixtures of dyes (e.g. Chapter 4, Section 4.1.2; Chapter 7, Section
7.1) and immunohistochemistry (Chapter 19, Section 19.12.2). Fixation and other pre-treatments
are often beyond the control of knowledgeable laboratory staff required to conduct histological
and histochemical investigations as a service to clinicians and researchers.
The particular hazards of histological processing (mainly toxicity and fire) are noted as they arise,
but this is not a textbook of laboratory safety, and the warnings do not cover every risk. Note
that in some institutions the use of certain chemicals may be forbidden because of real or (more
frequently) suspected hazards. It is necessary to comply with such prohibitions even if you do
not agree with them. Local regulations must also be followed for disposing of solvents and other
chemicals, and of materials of human or other biological origin.
What’s new in the fifth edition?
As with the fourth edition (2008) I have tried to include some newer procedures that seem likely
to become ‘standard’ methods in research, diagnostic pathology, or the preparation of teaching
viii Preface to the fifth edition
materials. The book now includes more methods for microorganisms (mostly in Chapters 6
and 18) than previously. Methods for endogenous and exogenous pigments have been added in
Chapter 13. This chapter now also includes techniques for detecting calcium oxalate deposits and
for staining bone (red) and cartilage (blue) in transparent whole-mount preparations, a procedure
much used in the investigation of teratogenic effects of drugs and other substances. Explanations
of the rationales of many techniques have been revised in the light of recent research.
Methods for fixing and processing tissues continue to become more numerous and more diverse,
and the first four chapters of this edition contain descriptions of various newer reagents and
techniques. Ordinary staining with dyes is carried out as much as ever, and several methods,
not all of them new, have been added to Chapters 6, 7 and 8. The reader will also find enough
theoretical and practical information to make up combinations of staining procedures appropriate
to the needs of the moment.
All chapters have been updated and there are changes on most pages. There are also more deletions
than in previous revisions – mostly of old methods that now are seldom used – to make way for
the new material. Overall there is more that needs to be said than there was seven years ago.
The first three editions of Histological and Histochemical Methods (1981, 1990, 1999) had
questions at the end of each chapter, with answers at the end of the book. These were dropped
from the fourth (2008) edition, in the mistaken belief that readers did not want them, to free up
printed pages. With this fifth edition, an updated set of questions and answers is available on the
publisher’s website www.scionpublishing.com/HHM5.
JOHN A. KIERNAN
London, Ontario, Canada
Acknowledgements
Thanks are due to many people who have given advice and criticism over the years. Among present
and former colleagues at the University of Western Ontario (UWO), I thank Kim Baines, Robert
C. Buck, M. George Cherian, Brian A. Flumerfelt, Peter Haase, Elizabeth A. Heinicke, Peeyush
K. Lala, Don Montemurro, Chris Naus, N. Rajakumar, Kem Rogers and the late Ted Walker. I
have also learned much from Graeme Berlyn (New Haven, CT), Charles Churukian (Rochester,
NY, deceased), Richard W. Dapson (Richland, MI), Chad Fagan (Rochester, NY), William Grizzle
(Birmingham, AB), Tony Henwood (Sydney, NSW), Richard W. Horobin (Sheffield, UK), David
P. Penney (Rochester, NY), Philip E. Reid (Vancouver, BC, deceased), Clive R. Taylor (Pasadena,
CA) and Dietrich Wittekind (Freiburg, Germany, deceased). Discussions over the internet have
taught me about histological and microscopical practices in many parts of the world. For sharing
their wisdom I thank several people, most of whom I have never met in person, including Russ
Allison (deceased), René Buesa, Gayle Callis, Freida Carson, Jim Elsam, Bryan Hewlett, George
Kumar, Ian Montgomery, Phil Oshel, Bob Richmond, Barry Rittman, Ron Stead and many others.
Comments and questions from graduate students at UWO have also prompted corrections and
clarification in several places.
My interest in histochemistry and histotechnology developed from using the methods in research,
which has been financed by grants from several agencies, including the Medical Research Council,
the Ontario Thoracic Society, the Natural Sciences and Engineering Research Council, the Multiple
Sclerosis Society of Canada, the Amyotrophic Lateral Sclerosis Society of Canada, the Ontario
Association of Medical Laboratories and, most recently, by the Biological Stain Commission, an
organization with important functions that are summarized in Chapter 5, Section 5.6.2.
Finally, I thank Dr Jonathan Ray of Scion Publishing for his guidance during the preparation of the
fourth and fifth editions, and his editorial staff for making corrections and improvements.
JOHN A. KIERNAN
London, Ontario, Canada