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Immunohistochemistry and Immunocytochemistry: Essential Methods, 2nd Edition



Immunohistochemistry and Immunocytochemistry: Essential Methods, 2nd Edition

Simon Renshaw (Editor)

ISBN: 978-1-118-71774-5 February 2017 Wiley-Blackwell 248 Pages



Immunohistochemistry and immunocytochemistry are invaluable tools for the visualization of tissue and cellular antigens in diagnostic and biological research environments. The need to obtain accurate, reliable and reproducible results is paramount.

It is with this fundamental aim in mind that we have compiled Immunohistochemistry: Essential Methods. We have achieved this by examining each aspect of immunochemistry in turn, with each chapter including detailed information regarding the subject matter in question. Each chapter is written by an expert in their field and includes protocols that are typically used in their own research. Subjects covered are, amongst others, antibodies and their production; selection of reporter labels; immunochemical staining methods and experimental design (both using single and multiple reporter labels); quality assurance; automated immunochemistry; confocal microscopy and electron microscopy. In addition, benefits and limitations of each approach are discussed within the chapters.
List of Contributors vii

Preface ix

Acknowledgements xi

Chapter 1: Antibodies for Immunochemistry 1
Mark Cooper and Sheriden Lummas

Introduction 1

Immunogens for Antibody Production 5

Antibody Production 12

Antibody Purification 16

Fragment Antibody Preparations 20

Antibody Labelling 21

Antibody Stability and Storage 23

References 24

Chapter 2: The Selection of Reporter Labels 25
Judith Langenick

Introduction 25

Enzymatic Labels 26

Fluorescence Detection 29

References 32

Chapter 3: Immunohistochemistry and Immunocytochemistry 35
Simon Renshaw

Specimen Formats for Immunochemistry 36

Fixation 37

Processing Tissue Blocks to Paraffin Wax 46

Microtomy 47

Tissue Microarrays 47

Specimen Storage 48

Decalcification 49

Antigen Retrieval 50

Controls 56

Immunochemical Staining Techniques (Optimizing a New Antibody) 57

Counterstains 71

Mounting 74

Troubleshooting 76

Examples of Immunostaining Photomicrographs 76

Acknowledgements 101

References 101

Chapter 4: Multiple Immunochemical Staining Techniques 103
Sofia Koch

Introduction 103

Methods and Approaches 115

References 122

Chapter 5: Quality Assurance in Immunochemistry 123
Peter Jackson and Michael Gandy

Introduction 123

Methods and Approaches 125

Automated Immunochemical Staining 147

Troubleshooting 149

References 154

Chapter 6: Automated Immunochemistry 157
Emanuel Schenck and Simon Renshaw

Introduction 157

Methods and Approaches 160

Other Forms of Automation 164

References 168

Chapter 7: Confocal Microscopy 169
Ann Wheeler

Introduction 169

When Should Confocal be Used? 173

Applications: For Example Co-localization, Quantification, 3D Visualization and Kinetics 173

How To Set Up a Confocal Experiment? 174

References 198

Further Readings 198

Chapter 8: Ultrastructural Immunochemistry 199
Jeremy Skepper and Janet Powell

Introduction 199

Methods and Approaches 207

References 222

Index 227