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PAT H OLOGY Education Guide Immunohistochemical Staining Methods Fifth Edition Education Guide | Immunohistochemical (IHC) Staining Methods Updated and Expanded Fifth Edition Editors J. Paul Robinson PhD and Jennifer Sturgis BS George L Kumar PhD Purdue University Cytometry Laboratory Purdue University | West Lafayette, IN, USA Managing Editor / Scientific Communications Manager Dako North America, Inc. | Carpinteria, CA 93013, USA Lars Rudbeck PhD Scientific Editor Dako Denmark A/S | Glostrup, Denmark Contributors Thomas Boenisch MS Retired Dako Consultant | Santa Barbara, CA, USA Clive R. Taylor MD, DPhil, FRCPath, FRCP (Ir) Department of Pathology and Laboratory Medicine University of Southern California | Los Angeles, CA, USA A.J. Farmilo PhD Independent Consultant | Canada (Formerly with Dako Canada) Ronald H. Stead PhD The Holburn Group of Companies Bowmanville, Ontario, Canada James F. Happel DLM (ASCP), HTL Massachusetts General Hospital | Boston, MA, USA Rashmil Saxena BFA, HT (ASCP)CM and Sunil Badve MD, FRCPath Clarian Pathology Laboratory Department of Pathology & Laboratory Medicine Indiana University | Indianapolis, IN, USA Marc Key PhD Celerus Diagnostics, Inc. | Carpinteria, CA, USA (Formerly with Dako) Lars Rudbeck PhD, Henrik Winther PhD, Sussie Steen Jensen MS, Sven Müller PhD, Steen H. Matthiesen PhD, Kirsten V. Nielsen MS, Ulla Henriksen PhD, Andreas Schönau MS, EBA, Nanna K. Christensen PhD, Helle Grann Wendelboe MS and Ole Feldballe Rasmussen PhD Dako Denmark A/S | Glostrup, Denmark Lars Winther PhD Independent consultant | Copenhagen Area, Denmark (Formerly with Dako Denmark) Dan Osborn Omega Optical, Inc | Brattleboro, VT, USA Robert M. Zucker PhD U.S. Environmental Protection Agency National Health and Environmental Effects Research Laboratory | Research Triangle Park, NC, USA Kirsten Bisgaard BSc Statens Serum Institut | Copenhagen, Denmark (Formerly with Dako Denmark) George L Kumar PhD, Gale E. Pace BS, Joachim Schmid PhD and Mark Verardo PhD, Dako North America | Carpinteria, CA, USA David C. Spaulding, PhD Stellar Biotechnologies | Port Hueneme, CA, USA Karen N. Atwood BS, MT (ASCP), CLS Retired, Dako North America | Carpinteria, CA, USA © Copyright 2009 Dako North America, Carpinteria, California. All rights reserved. No part of this book may be reproduced, copied or transmitted without written permission. US $50 IHC Staining Methods, Fifth Edition | i ii | IHC Staining Methods, Fifth Edition Table of Contents Preface Chapter 1 | Antibodies 1 Thomas Boenisch MS (Revised by Betsy Spaulding, Henrik Winther PhD and Sussie Steen Jensen) Immunoglobulins | IgG | IgM | Polyclonal Antibodies | Monoclonal Antibodies | Polyclonal Antibodies vs. Monoclonal Antibodies | Antibody Affinity | Antibody Cross-Reactivity | Antibody Reaction Rates | Antibody Stability | Handling of Antibodies | Receiving | Storage | Storage Containers | Storage Temperature | Use and Care Chapter 2 | Basic Immunochemistry 11 Thomas Boenisch MS Antibody Titer | Antibody Dilution | Antibody Incubation | Incubation Time | Incubation Temperature Chapter 3 | Basic Enzymology 15 Thomas Boenisch MS Enzymes | Suggested Procedures for Substrate-Cromogen Reagents Chapter 4 | Immunohistochemical Standardization and Ready-to-Use Antibodies 21 Clive R. Taylor MD, DPhil, FRCPath, FRCP (Ir) Introduction and Background: The Growing Consensus for ‘Standardization’ | Ready-to-Use (RTU) Reagents | Reagent Validation | Primary Antibody, Selection, Validation, and Optimization | Secondary or Labeling Reagents; Protocols | Controls | Impact of RTUs on Other Aspects of the Total T