Immunohistochemistry and Immunocytochemistry (eBook)

Essential Methods

Simon Renshaw (Herausgeber)

eBook Download: PDF

2017 | 2. Auflage
John Wiley & Sons (Verlag)
978-1-118-71775-2 (ISBN)

Infos

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.

Simon Renshaw is the editor of Immunohistochemistry and Immunocytochemistry: Essential Methods, 2nd Edition, published by Wiley.

Inhalt

Cover 1
Title Page 5
Copyright 6
Contents 7
List of Contributors 9
Preface 11
Acknowledgements 13
Chapter 1 Antibodies for Immunochemistry 15
Introduction 15
Typical Antibody Structure 16
Antibody Structure Is Optimized for Its Function 18
Immunogens for Antibody Production 19
Protein Structure Considerations for Antibody Generation 19
Types of Immunogens for Antibody Production 20
Epitope Prediction Tools 20
Considerations for Peptide Immunogen Design 21
Peptide Carrier Protein 24
Conjugating Peptide Immunogens to Carrier Protein 25
Antibody Production 26
Polyclonal Antibody Production 26
Monoclonal Antibody Production 28
Monoclonal and Polyclonal Antibodies for Immunochemistry 29
Antibody Purification 30
Ammonium Sulphate Precipitation 30
Affinity Purification 30
Proteins A and G Resins 31
Protein L 32
Ion Exchange 33
NHS and Iodoacetyl-Activated Resins 33
Fragment Antibody Preparations 34
Antibody Labelling 35
Fluorescent Reporters 36
Enzyme Reporters 37
Antibody Stability and Storage 37
References 38
Chapter 2 The Selection of Reporter Labels 39
Introduction 39
Enzymatic Labels 40
HRP 41
AP 42
Glucose Oxidase and Beta-Galactosidase (B-GAL) 43
Fluorescence Detection 43
Fluorochrome Characteristics 44
References 46
Chapter 3 Immunohistochemistry and Immunocytochemistry 49
Specimen Formats for Immunochemistry 50
Paraffin Embedded 50
Frozen 50
Free Floating 51
Cytological 51
Fixation 51
Types of Fixatives 52
Other Beneficial Effects of Fixation 58
Quality Control Considerations Regarding Fixation 58
Processing Tissue Blocks to Paraffin Wax 60
Microtomy 61
Tissue Microarrays 61
Specimen Storage 62
Paraffin-Embedded Tissue Sections 62
Frozen Tissue Sections 63
Cytological Specimens 63
Fluorescently Labelled Specimens 63
Decalcification 63
Antigen Retrieval 64
Heat-Induced Antigen Retrieval 65
Enzymatic Antigen Retrieval 68
Controls 70
Antigen Controls 70
Reagent Controls 70
Immunochemical Staining Techniques (Optimizing a New Antibody) 71
Pre-Antibody Purchase/Optimization Research 71
Formulating an Antibody Optimization Experiment 73
Choice of Detection System 75
Immunostaining Techniques 78
Counterstains 85
Counterstains for Enzyme/Chromogen Immunostaining 85
Counterstains for Fluorescent Immunostaining 86
Mounting 88
Troubleshooting 90
Examples of Immunostaining Photomicrographs 90
Immunocytochemistry/Immunofluorescence 91
Immunohistochemistry 104
Acknowledgements 115
References 115
Chapter 4 Multiple Immunochemical Staining Techniques 117
Introduction 117
Advantages and Technical Challenges 117
Multiple Immunochemical Staining Method Selection 118
Designing a Multiple Immunochemical Staining Experiment 120
Double Staining Using Same-Species Primary Antibodies 126
Methods and Approaches 129
References 136
Chapter 5 Quality Assurance in Immunochemistry 137
Introduction 137
Methods and Approaches 139
Fixation and Tissue Processing 139
Microtomy 142
Decalcification 143
Antigen Retrieval 144
Immunochemical Methodologies 148
Controls 150
Microscopic Interpretation 153
Background Staining 154
Automated Immunochemical Staining 161
Artefacts of Fully Automated Staining 162
Troubleshooting 163
References 168
Chapter 6 Automated Immunochemistry 171
Introduction 171
Defining the Needs 172
Overview of Automated Platforms for Immunochemical Staining 173
System Contrasts 173
Methods and Approaches 174
Overall Configuration of the System 174
Reagent Application Method 175
Ability of the Instrument to Apply Heat to Individual Slides within a Narrow Temperature Range 176
Other Special Features 176
System Running Costs 177
System Failure Safeguards 177
Other Forms of Automation 178
Automated Antigen Retrieval Systems 178
Digital Pathology 179
References 182
Chapter 7 Confocal Microscopy 183
Introduction 183
History of Confocal 184
Widefield versus Confocal Microscopy 184
How a Confocal Works? 185
When Should Confocal be Used? 187
Applications: for Example Co-Localization, Quantification, 3D Visualization and Kinetics 187
Limitations of Confocal Microscopy: Depth Penetration and Resolution 188
How to Set Up a Confocal Experiment? 188
Visualization of the Sample on the Confocal 188
Setting Up a Microscope for Confocal Microscopy 189
Selecting the Excitation and Emission Pathways 190
Multicolour Confocal Imaging for Co-localization 191
Optimizing the Detectors 194
Three-Dimensional (3D) Confocal Imaging of a Volume 197
Collecting Larger Samples Using Confocal Tiling 200
Live Cell Imaging 200
Pros and Cons of Point Scanning for Live Imaging 202
Fast Live Imaging 205
Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Photoactivation (PA) 208
Confocal Image processing 209
Image Processing for Presentation 209
Quantification of Confocal Images 211
Deconvolution of Confocal Images 211
Presentation of Confocal Images in 3D 211
References 212
Further Readings 212
Chapter 8 Ultrastructural Immunochemistry 213
Introduction 213
Fixation and Its Effect on Antigen-Antibody Binding 214
Controls 218
Why Do We Need to Use Electron Microscopy? 218
Quantification 219
Methods and Approaches 221
Epoxy Resin Section 221
The Acrylic Resins London Resin (LR) White and Gold 222
Freeze Substitution and Low-Temperature Embedding in Lowicryl HM20 225
Ultrathin-Thawed Cryosections 225
New Developments 228
Recommended Protocols 229
References 236
Index 241
EULA 250

Produkt-Details

• Erscheint lt. Verlag: 15.2.2017
• Reihe/Serie: Essential Methods
• Reihe/Serie: Essential Methods
• Reihe/Serie: Essential Methods
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete
• Themenwelt: Studium ► 2. Studienabschnitt (Klinik) ► Anamnese / Körperliche Untersuchung
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Mikrobiologie / Immunologie
• Themenwelt: Technik
• Schlagworte: Biowissenschaften • Cell & Molecular Biology • Chemie • Chemistry • Immunchemie • immunochemistry • Immunohistochemistry, Immunocytochemistry, Immunofluorescence, IHC, ICC, IF, Antibody, Antibodies, Immunoassay, Histology • Life Sciences • Microscopy • Mikroskopie • Zell- u. Molekularbiologie
• ISBN-10: 1-118-71775-9 / 1118717759
• ISBN-13: 978-1-118-71775-2 / 9781118717752