Genome Visualization by Classic Methods in Light Microscopy PDF
Preview Genome Visualization by Classic Methods in Light Microscopy
GENOME 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 Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2001 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20140807 International Standard Book Number-13: 978-1-4200-4251-1 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the valid- ity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or uti- lized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopy- ing, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com 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 V 0043fr_m.fm Page VI Tuesday, October 10, 2000 2:18 PM 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 0043fr_m.fm Page VIII Tuesday, October 10, 2000 2:18 PM 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 0043fr_m.fm Page IX Tuesday, October 10, 2000 2:18 PM 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
The list of books you might like
