Immunohistochemistry: Basics and Methods

Immunohistochemistry: Basics and Methods

Igor B. Buchwalow l Werner Böcker Immunohistochemistry: Basics and Methods

Prof. Dr. Igor B. Buchwalow Prof. Dr. Werner Böcker Gerhard-Domagk-Institut für Pathologie Domagkstr. 17 48149 Münster Germany New addresses: Prof. Dr. Igor B. Buchwalow Institute for Hematopathology Fangdieckstrasse 75a 22547 Hamburg Germany buchwalow@pathologie-hh.de; buchwalow@hotmail.de Prof. Dr. Werner Böcker Consultation and Reference Center for Gyneco- and Breast-Pathology Fangdieckstrasse 75a 22547 Hamburg Germany boecker@pathologie-hh.de; werner.boecker@gmail.com ISBN: 978-3-642-04608-7 e-isbn: 978-3-642-04609-4 DOI 10.1007/978-3-642-04609-4 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009938017 # Springer-Verlag Berlin Heidelberg 2010 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, reuse 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. Violations are liable to prosecution under the German Copyright Law. 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. Cover Illustration: Double immunolabeling of cytokeratins in a duct wall of the human mammary gland, see Chap. 8, Fig. 3 (photo by Igor B. Buchwalow, Gerhard-Domagk-Institut für Pathologie, Münster, Germany) Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface Combining two different scientific disciplines morphology and immunochemistry immunohistochemistry has developed as an important instrument in research and clinical pathology. A basic understanding of underlying principles and potential problems is unavoidable if you want to be successful in your use of immunohistochemistry, as well as in getting your papers published and your research grants funded. While many excellent texts and monographs exist which cover various aspects of immunohistochemistry, the lack of a concise comprehensive guide to using these methods was a major motivation for writing this book. Our intention was to create an easy-to-read and focused resource based on state-of-the-art information for a broad audience ranging from students and technical assistants to experienced researchers. This handbook has a concise format, with protocols and instructions for methods immediately following the short introductory theoretical material in each chapter. Being conscious of the growing role of Internet as an information source, we have found it reasonable in many cases to substitute citing books and journal publications with corresponding Internet websites. Where possible, commercial sources of reagents, kits, and equipment are listed throughout the text instead of in a separate index. Though each chapter is small and introductory, this handbook itself is self-sufficient and provides a comprehensive look at the principles of immunohistochemistry. For readers wanting further depth of knowledge, each chapter is backed up by a short list of carefully selected original articles. During the last decade, pioneering efforts of histochemists have led to an immense improvement in the reagents and protocols. The researcher is urged always to determine the reason for every method and step before doing it. This handbook is intended to help readers to avoid troubles in the choice of an adequate method, which happens when using standard textbooks. For this handbook, we carefully selected established methods and easy-to-adopt protocols, paying attention to modern developments in immunohistochemistry, such as antigen retrieval, signal amplification, the use of epitope tags in immunohistochemistry, multiple immunolabeling or diagnostic immunohistochemistry. Each of the methods described in this handbook v

vi Preface was proved by the authors; many of these methods are routinely used in daily practice in their institute. All the practical methods advocated are clearly described, with accompanying tables, and the results obtainable are illustrated with colour micrographs. Acknowledgements We thank Vera Samoilova for the perfect technical assistance and other colleagues from the Münster University Clinic for sharing probes and reagents. Igor B. Buchwalow and Werner Böcker Münster

Contents 1 Antibodies for Immunohistochemistry... 1 1.1 Structure of Antibodies... 2 1.2 Polyclonal Antibodies... 4 1.3 Mouse Monoclonal Antibodies... 4 1.4 Rabbit Monoclonal Antibodies... 5 1.5 Protein A and Protein G in Immunohistochemistry... 7 References..... 8 2 Antibody Labeling and the Choice of the Label... 9 2.1 Covalent Labeling of Antibodies... 9 2.2 Non-Covalent Labeling of Primary Antibodies with Labeled Fab Fragments... 10 2.3 Enzyme Labels for Light Microscopy... 13 2.4 Fluorophore Labels for Fluorescence Microscopy... 15 2.5 Colloidal Gold Labels for Electron Microscopy... 16 References..... 17 3 Probes Processing in Immunohistochemistry... 19 3.1 Fixation in Immunohistochemistry... 19 3.1.1 Fixation in Alcohols and Acetone... 20 3.1.2 Fixation in Formaldehyde... 20 3.1.3 Effect of Formaldehyde Fixation on Antigen Antibody Binding... 21 3.2 Paraffin Sections for Immunohistochemical Analysis... 22 3.2.1 Embedding and Cutting... 22 3.2.2 Mounting Paraffin Sections onto Slides... 23 3.3 Cryosections for Immunohistochemical Analysis... 24 3.4 Buffers for Washing and Antibody Dilution.... 25 3.5 Mounting Following Immunohistochemical Staining... 27 3.6 Storage Following Immunohistochemical Staining... 28 References..... 28 vii

viii Contents 4 Working with Antibodies... 31 4.1 Direct Immunostaining Method... 31 4.2 Indirect Immunostaining Method... 33 4.3 The Choice of Antibodies... 35 4.3.1 The Choice of Primary Antibodies... 35 4.3.2 The Choice of Secondary Antibodies.... 36 4.4 Optimal Concentration of the Antibody... 37 4.5 Specificity Controls in Immunohistochemistry... 38 References..... 39 5 Background Staining, Autofluorescence and Blocking Steps... 41 5.1 Fc Receptors... 41 5.2 Endogenous Peroxidase... 42 5.3 Endogenous Alkaline Phosphatase... 43 5.4 Endogenous Biotin... 43 5.5 Autofluorescence or The Wood Through the Trees... 44 References..... 46 6 Immunostaining Enhancement... 47 6.1 Antigen Retrieval... 47 6.1.1 Heat-Induced Antigen Retrieval... 48 6.1.2 Proteolytic Antigen Retrieval... 50 6.2 Signal Amplification... 51 6.2.1 Avidin Biotin Complex... 52 6.2.2 Chain Polymer-Conjugated Technology... 53 6.2.3 Tyramide Signal Amplification... 54 6.2.4 Amplification of the Amplifier... 58 References..... 58 7 Multiple Multicolor Immunoenzyme Staining... 61 7.1 Simultaneous Immunoenzymatic Double Staining... 61 7.2 Sequential Immunoenzymatic Double/Multiple Staining... 63 7.3 Stripping Buffers for Sequential Immunoenzymatic Double Staining... 65 7.4 Nuclear Counterstaining Following Immunoenzyme Labeling... 66 References..... 66 8 Multiple Immunofluorescence Staining... 69 8.1 Double Immunofluorescence Indirect Staining Using Primary Antibodies Raised in Two Different Host Species... 70 8.2 Using Primary Antibodies of Different IgG Isotype... 71 8.3 Using Haptenylated Primary Antibodies... 74 References..... 76

Contents ix 9 Antigen Detection on Tissues Using Primary Antibody Raised in the Same Species... 77 9.1 Haptenylation of Primary Antibodies with the Following Use of Secondary Antibodies Recognizing the Corresponding Hapten... 78 9.2 Blocking Endogenous Tissue Immunoglobulins Homologous to Primary Antibody by Preincubation with Unconjugated Fab Fragments... 79 References..... 81 10 Probes for Staining Specific Cellular Organelles... 83 10.1 Nuclear Markers... 83 10.1.1 Nuclear Fluorescent Counterstaining... 83 10.1.2 Cell-Proliferation Nuclear Markers.... 85 10.1.3 Nuclear Envelope Markers... 85 10.1.4 Nucleolar Markers... 86 10.2 Probes for Mitochondria... 87 10.3 Probes for Endoplasmic Reticulum and Golgi Apparatus... 88 10.4 Endocytic Pathways... 89 10.5 Probes for Cytoskeleton... 91 10.6 Phalloidin Probes for Actin Filaments... 92 References..... 94 11 The Use of Epitope Tags in Histochemistry... 95 References..... 98 12 Immunohistochemistry at the Ultrastructural Level... 99 12.1 Colloidal Gold Conjugates... 99 12.2 Fixation for Ultrastructural Immunohistochemistry... 100 12.3 Resin Etching and Heat-Induced Antigen Unmasking in Resin Sections... 101 12.4 Immunogold Labeling Procedure... 104 References..... 107 13 Diagnostic Immunohistochemistry... 109 13.1 Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast... 110 13.1.1 Human Keratins and Their Expression Patterns in Breast Epithelium... 110 13.1.2 Progenitor Cell Keratins K5/K14...... 110 13.1.3 K17: Keratin of Basal/Myoepithelial Cells... 112 13.1.4 Glandular Keratins... 113 13.1.5 K1/K10: Major Keratins of Keratinocyte Differentiation and Keratinization..... 114 13.1.6 Myoepithelial Differentiation Markers... 115

x Contents 13.1.7 Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast... 117 13.1.8 Carcinomas of Luminal Phenotype (Luminal Type Carcinomas) in the Breast... 117 13.1.9 Tumors of Basal Phenotype... 118 13.1.10 Squamous Cell Carcinomas... 119 13.2 Tissue Microarrays... 122 References..... 125 14 A Picture Is Worth a Thousand Words... 129 14.1 Brightfield Microscopy... 129 14.1.1 How to Set Up a Light Microscope Properly for Transmitted Light Illumination: Köhler Illumination... 130 14.1.2 The Choice of Microscope Objectives... 131 14.2 Fluorescence Microscopy... 134 14.2.1 The Choice of Filter Cubes for Fluorescence Microscopy... 134 14.2.2 The Choice of Fluorophores for Multiple Immunostaining... 137 References..... 139 Glossary... 141 Index... 151