EDUCATIONAL IHC Guidebook Sixth Edition Education Guide Immunohistochemical Staining Methods Immunohistochemical Staining Methods – Sixth Edition Corporate Headquarters Australia Canada France Japan Poland United Kingdom Denmark +61 3 9357 0892 +1 905 335 3256 +33 1 64 53 61 44 +81 3 5802 7211 +48 58 661 1879 +44 (0)1 353 66 99 11 +45 44 85 95 00 Austria China Germany Korea Spain United States of America +43 1 408 43 34 0 +86 21 3612 7091 +49 40 69 69 470 +82 2 402 6775 +34 93 499 05 06 +1 805 566 6655 Belgium Denmark Ireland The Netherlands Sweden www.dako.com +32 (0) 16 38 72 20 +45 44 85 97 56 +353 1 479 0568 +31 20 42 11 100 +46 8 556 20 600 Represented in more Brazil Finland Italy Norway Switzerland than 100 countries +55 11 50708300 +358 9 348 73 950 +39 02 58 078 1 +47 23 14 05 40 +41 41 760 11 66 08002 07NOV13 Education Guide Immunohistochemical Staining Methods Sixth Edition Editors Clive R. Taylor, MD, D.Phil, Past Chair, Department of Pathology, University of Southern California; Past Council Member and President, Association of Pathology Chairs Lars Rudbeck, PhD, MSc, Scientific Editor, Dako Denmark Graphic Design Anja H. Sjørup, MSc, Graphic Designer, Dako Denmark © Copyright 2013 | Dako Denmark A/S, An Agilent Technologies Company. All rights reserved. No part of this book may be reproduced, copied or transmitted without written permission. Immunohistochemical Staining Methods | Table of Contents Table of Contents Immunohistochemical Staining Methods Part I: The Staining Process Preface.................................................................................................. 8 Chapter 1 | Introduction to Immunohistochemistry . 10 by C.R. Taylor 1.1 | Introduction.................................................................................. 11 1.2 | History of Immunohistochemistry . 13 1.3 | Standardization in Clinical Immunochemistry vs. Immunohistochemistry ..................15 1.4 | Growing Consensus for Standardization . 16 1.5 | Standardization Starts in the Surgery Room . 16 1.6 | Future Aspects for Standardization of Immunohistochemistry .....................................17 Chapter 2 | Fixation and Other Pre-Analytical Factors . 20 by E.C. Colley & R.H. Stead 2.1 | Introduction.................................................................................. 21 2.2 | Tissue Handling . 21 2.3 | Fixation . 23 2.4 | Tissue and Slide Processing . 25 2.5 | Special Tissue Preparations ..................................................... 26 2.6 | Control Material ........................................................................... 27 2.7 | Validation...................................................................................... 28 2.8 | Conclusions . 29 Chapter 3 | Antigen Retrieval . .30 by S-R Shi & C.R. Taylor 3.1 | Introduction.................................................................................. 31 3.2 | Major Factors that Influence the Effect of Antigen Retrieval .........................................32 3.3 | Standardization of AR – The “Test Battery” Approach . 34 3.4 | Application of AR Techniques – The Basic Principles . 36 3.5 | AR-IHC-based Research and Diagnostics . 36 3.6 | Reagents and Protocols ............................................................ 36 3.7 | Water Bath Methods................................................................... 36 3.8 | Pressure Cooker Heating .......................................................... 38 3.9 | Autoclave Heating ...................................................................... 39 3.10 | Microwave Oven Heating . 39 3.11 | Proteolytic Pre-treatment......................................................... 40 3.12 | Combined Proteolytic Pre-treatment and Heat-Induced Antigen Retrieval..............40 3.13 | Combined Deparaffinization and Antigen Retrieval . 41 3.14 | Conclusions............................................................................... 42 Table of Contents | Immunohistochemical Staining Methods Chapter 4 | Selection of the Primary Antibody . 46 by S. Nielsen 4.1 | Introduction.................................................................................. 47 4.2 | Seletion of the Proper Antibody . 47 4.3 | A New (Replacement) Antibody for an Old Marker . 49 4.4 | A New Antibody for New Marker in the Pathology Laboratory .....................................51 4.5 | A New Experiment in the Pathology Laboratory . 52 4.6 | Examples of Good and Poor Antibodies . 54 4.7 | Current and Future Challenges................................................ 57 Chapter 5 | Staining Protocol Optimization . 60 by L. Jacobsen, M. Nielsen, S. Månsson & L. Rudbeck 5.1 | Introduction.................................................................................. 61 5.2 | The Basis for an Optimal Staining Protocol . 62 5.3 | The protocol................................................................................. 64 5.4 | Reproducibility of IHC................................................................ 70 5.5 | Requirements for Controls . 70 5.6 | Verification and Validation of a Protocol . 73 5.7 | Guide to the Development of an Optimal Staining Protocol . 73 5.8 | Protocol Performance for Abnormal Tissue . 73 5.9 | Concluding remarks................................................................... 76 Chapter 6 | Immunohistochemistry Staining Methods . 78 by K. Petersen & H.C. Pedersen 6.1 | Introduction.................................................................................. 79 6.2 | Avidin-Biotin Immunohistochemistry . 79 6.3 | Polymer-Based Immunohistochemistry . 80 6.4 | Catalysed Signal Amplification (CSA) . 81 6.5 | Fluorescyl-tyramide Amplification . 82 6.6 | Improved Catalysed Signal Amplification (iCSA) . 82 6.7 | Multi-Staining Immunohistochemistry . ..