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Full Description
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.
Contents
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
