Table Of ContentGENOME
VISUALIZATION
by CLASSIC METHODS
in LIGHT
MICROSCOPY
Methods in Visualization
Series Editor: Gérard Morel
In Situ Hybridization in Light Microscopy
Gérard Morel and Annie Cavalier
Visualization of Receptors: In Situ Applications
of Radioligand Binding
Emmanuel Moyse and Slavica M. Krantic
Genome Visualization by Classic Methods
in Light Microscopy
Jean-Marie Exbrayat
Imaging of Nucleic Acids and Quantification
in Phototonic Microscopy
Xavier Ronot and Yves Usson
In Situ Hybridization in Electron Microscopy
Gérard Morel, Annie Cavalier,
and Lynda Williams
GENOME
VISUALIZATION
by CLASSIC METHODS
in LIGHT
MICROSCOPY
Jean-Marie Exbrayat, Ph.D., D.Sc.
CRC Press
Boca Raton London New York Washington, D.C.
CRC Press
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© 2001 by Taylor & Francis Group, LLC
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Version Date: 20140807
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0043fr_m.fm Page V Tuesday, October 10, 2000 2:18 PM
SERIES PREFACE
Visualizing molecules inside organisms, tissues, or cells continues to be an exciting challenge for
cell biologists. With new discoveries in physics, chemistry, immunology, pharmacology, molecular
biology, analytical methods, etc., limits and possibilities are expanded, not only for older visualizing
methods (photonic and electronic microscopy), but also for more recent methods (confocal and
scanning tunneling microscopy). These visualization techniques have gained so much in specificity
and sensitivity that many researchers are considering expansion from in-tube to in situ experiments.
The application potentials are expanding not only in pathology applications but also in more
restricted applications such as tridimensional structural analysis or functional genomics.
This series addresses the need for information in this field by presenting theoretical and
technical information on a wide variety of related subjects: in situ techniques, visualization of
structures, localization and interaction of molecules, functional dynamism in vitro or in vivo.
The tasks involved in developing these methods often deter researchers and students from
using them. To overcome this, the techniques are presented with supporting materials such as
governing principles, sample preparation, data analysis, and carefully selected protocols. Addi-
tionally, at every step we insert guidelines, comments, and pointers on ways to increase sensitivity
and specificity, as well as to reduce background noise. Consistent throughout this series is an
original two-column presentation with conceptual schematics, synthesizing tables, and useful com-
ments that help the user to quickly locate protocols and identify limits of specific protocols within
the parameter being investigated.
The titles in this series are written by experts who provide to both newcomers and seasoned
researchers a theoretical and practical approach to cellular biology and empower them with tools
to develop or optimize protocols and to visualize their results. The series is useful to the experienced
histologist as well as to the student confronting identification or analytical expression problems. It
provides technical clues that could only be available through long-time research experience.
Gérard Morel, Ph.D.
Series Editor
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0043fr_m.fm Page VII Tuesday, October 10, 2000 2:18 PM
GENERAL INTRODUCTION
Visualization of nucleic acids has become The purpose of this book is to provide
indispensable to studying cells, tissues, and insight into several classic techniques, histolog-
organisms, to understanding development, dif- ical as well as histochemical, that can be used to
ferentiation, and physiology, and to studying appreciate the nucleic acid status of the cell as
pathological disorders. Visualization is also well as to provide an overview of RNA and
used to determine the effects of a pharmaceu- DNA distribution in cells and tissues, more or
tical or a toxic molecule on cells, and is often less precisely according to the information.
essential for routine examination in clinical ser- Some of the techniques are relatively easy to
vices. Appreciation of the expression of genes perform in a nonspecialized laboratory. Others
in a cell is also of interest in biotechnology. are more difficult. Certain techniques permit
To visualize nucleic acids, most tech- visualization and quantification of DNA and/or
niques use dyes of natural or synthetic origin. RNA distribution in tissues. Several of them
Although, the action of the dyes is not always permit choice of a more precise method to fur-
precisely known, their affinity and specificity ther explicate the genome.
are no longer in doubt. More precise methods Numerous methods for DNA and RNA visu-
are based upon true chemical reactions in situ alization exist, but few recent analytical books
in which a nucleic acid is one reagent and the on the topic are available. This book presents an
other is a dye molecule. In these histochemical analytical approach. This approach seems valu-
methods, physicochemical conditions are able based on the author’s participation at the
known. The results of these reactions must symposia organized by Dr. Gérard Morel, the
always be validated by means of a negative editor of the series of which this book is a part,
control reaction in which DNA or RNA is based on the author’s more than 20 years of
deleted by use of chemical or enzymatic teaching in a school of technicians specializing
hydrolysis. in histology, and on the daily use of histology
Today, researchers have access to for the author’s own research.
increasingly more precise and more selective The book is organized in eight chapters. In
methods for visualization. Immunocytochemi- the first, visualization principles are described.
cal methods now use an antibody–antigen reac- The second chapter concerns preparation of tis-
tion, where a nucleic acid is the antigenic sues; and the third covers staining by classic
molecule. With in situ hybridization, genes, dyes. In the fourth chapter, histochemical meth-
DNA, and RNA are precisely visualized with ods are described. The fifth chapter concerns
nucleic probes. At a time in which precision is fluorescent dyes. In the sixth chapter, observa-
the rule, precise quantification, aided by pow- tion phases are described, along with section
erful computers, is always indispensable in mounting and a presentation on the light micro-
tracking gene expression. Numerous methods scope. Preparation of products is given in Chap-
permit such analysis by flux cytometry, as well ter 7. In the eighth chapter, some protocols that
as by automatic quantitative image analysis. are used in the author’s laboratory are
In this age of genetics, nucleic acid visualiza- described. The book ends with a few examples
tion is a necessary part of many scientific of staining and a glossary.
investigations.
VII
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ACKNOWLEDGMENTS
The author thanks Dr. Gérard Morel, the series editor of Methods in Visualization, who gave
numerous suggestions for improving and expanding the manuscript. He also thanks Genevieve
Escudie, Jeanne Estabel, and Paulette Pujol, who have taught histology and cytology with him for
several years, and Marie-Therese Laurent, a skilled technician whose ability and effectiveness
continue to contribute significantly to the improvement of methods that are used in the author’s
laboratory.
VIII
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THE AUTHOR
Jean-Marie Exbrayat, Ph.D., D.Sc., is Professor at the Catholic University in Lyon (France) where
he manages the Laboratory of General Biology and the School of Histology. He is also Directeur
d'Etudes (Professor) at the Ecole Pratique des Hautes Etudes, where he manages the Laboratory
of Vertebrate Reproduction and Development.
Professor Exbrayat obtained his M.S and Ph.D. in 1974 and 1977, respectively, from the
Montpellier University (France). He was appointed assistant, then master assistant, of biology at
the Catholic University in Lyon in 1978, and became doctor of sciences in 1986. He became a
professor in 1987 and also Directeur d'Etudes (Professor) at the Ecole Pratique des Hautes Etudes
(Practical School of High Studies) in 1991.
He is a member of the New York Academy of Sciences, American Association for the
Advancement of Sciences, Societas Europaea Herpetologica, Zoological Society of France, Her-
petological Society of France (of which he was General Secretary from 1991 until 1997), and the
French Association of Histotechnology.
Professor Exbrayat is the author of more than 150 papers and four books. His current major
interests include the variations of genital tracts and endocrine organs during reproductive cycles,
as well as embryonic development in lower vertebrates.
IX
