Introduction to Immunocytochemistry

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ISBN: 9781859962084 | 1859962084
Cover: Nonspecific Binding
Copyright: 7/29/2003

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Now in its third edition, Introduction to Immunocytochemistry is an essential text for newcomers to the field, and will be a valuable reference for more experienced workers. Extensively expanded and updated, this volume covers both background theory and practical applications, including immunostaining methods using fluorescent, enzyme and gold labels.

Abbreviations

Preface

xiii

Key to symbols

Introduction

(4)

Definition

(1)

History and development

(2)

References

(2)

Production of Antibodies

(8)

Immunization

(1)

Testing

(1)

Region-specific antibodies

(2)

Monoclonal antibodies

(1)

Selection of antibodies by phage display

(1)

Characteristics of a `good' antibody

(1)

References

(2)

Preparation of Tissue for Immunocytochemistry

(32)

Fixation

(3)

Cross-linking fixatives

(1)

Precipitant fixatives

(1)

Combination fixatives

(1)

Fixed, paraffin-embedded tissue

(1)

Fresh material -- frozen sections and cell preparations

(4)

Frozen sections

(1)

Whole cell preparations

(2)

Pre-fixed, non-embedded tissue

(2)

Pre-fixed frozen sections

(1)

Pre-fixed Vibratome sections

(1)

Whole-mounts

(1)

Permeabilization

(1)

Freeze-dried tissue

(1)

Tissue storage

(2)

Paraffin blocks and sections

(1)

Frozen blocks and sections

(1)

Cell preparations

(1)

Adherence of sections and cell preparations to slides

(1)

Antigen retrieval in fixed tissues

(5)

Washing

(1)

Protease treatment

(2)

Heat-mediated antigen retrieval

(3)

References

(4)

Colour plate section

(12)

Visualizing the End-product of Reaction

(14)

Fluorescent labels

(3)

Advantages

(1)

Disadvantages

(1)

Uses of immunofluorescence

(1)

Fluorescein

(1)

Rhodamine

(1)

Phycoerythrin

(1)

AMCA

(1)

Other fluorophores

(1)

Fluorescent counterstains

(1)

Enzyme labels

(4)

Peroxidase

(3)

Alkaline phosphatase

(1)

Glucose oxidase

(1)

β-D-Galactosidase

(1)

Gold labels

(2)

Colloidal gold

(1)

Nanogold

(1)

Other labels

(1)

Biotin

(1)

Haptens

(1)

Radioisotopes

(1)

References

(3)

Non-specific Staining due to Tissue Factors

(4)

Causes of non-specific binding

(1)

Charged sites

(1)

Hydrophobic attraction

(1)

Fc receptors

(1)

Prevention of non-specific binding

(1)

Other problems

(1)

Endogenous enzymes

(1)

Endogenous biotin

(1)

Autofluorescence

(1)

References

(1)

Methods

(18)

General considerations

(4)

Buffers

(1)

Antibody diluent and storage

(1)

Antibody dilution relative to reaction time, temperature and technique

(2)

Methods

(12)

Nature of antibodies (IgG)

(2)

Application of antibodies to preparations

(1)

Direct (one-step) method

(2)

Indirect (two-step) method

(1)

Three-layer methods

(3)

Avidin--biotin methods

(3)

References

(2)

Testing Antibodies: Specificity and Essential Controls

(12)

Testing a new primary antibody

(5)

A primary antibody with a known localization

(4)

A primary antibody with an unknown localization

(1)

Negative control for polyclonal antibodies -- normal serum

(1)

Negative controls for monoclonal antibodies

(1)

Testing for non-specific binding of second and third reagents

(1)

Non-specific or unwanted specific staining due to antibody factors

(3)

Unwanted specific staining of unknown antigens

(1)

Non-specific binding of antisera to basic proteins

(1)

Unwanted specific cross-reactivity of anti-immunoglobulins

(1)

Cross-reactivity of the primary antibody with related antigens

(2)

Remedies for non-specificity due to tissue factors

(1)

Blocking binding sites with normal serum

(1)

Absorption with tissue powder

(1)

Remedies for non-specificity due to heterogeneity of the antibody

(1)

Dilution

(1)

Affinity purification

(1)

Remedies for non-specificity due to cross-reactivity

(1)

Essential staining controls

(1)

Negative controls

(1)

Positive controls

(1)

Experimental controls

(1)

References

(2)

Increasing Sensitivity and Enhancing Standard Methods

(10)

Increasing sensitivity

(7)

Immunogold with silver enhancement

(1)

Build-up methods

(3)

Tyramine signal amplification (TSA)

(3)

Intensification of the peroxidase/DAB/H2O2 product

100

(1)

Post-reaction intensification

100

(1)

Intensification during the peroxidase reaction

101

(1)

References

101

(2)

Multiple Immunostaining

103

(12)

Double direct immunostaining with separately labelled primary antibodies

103

(1)

Double immunostaining with primary antibodies raised in different species, or of different immunoglobulin sub-class

104

(2)

Double immunoenzymatic method

104

(2)

Double immunofluorescence method

106

(1)

Triple immunostaining

106

(1)

Unlabelled primary antibodies from the same species

106

(7)

The problem

106

(1)

Elution methods

107

(2)

Indirect double immunostaining without elution

109

(4)

References

113

(2)

Immunocytochemistry for the Transmission Electron Microscope

115

(10)

Principles

115

(1)

Fixation

115

(2)

Pre-embedding immunocytochemistry

116

(1)

Non-embedding immunocytochemistry

116

(1)

Processing to resin

117

(1)

Labels

118

(1)

Sectioning resin blocks

119

(1)

Pre-treatment

120

(1)

Immunolabelling procedure

121

(1)

Immunolabelling with peroxidase

121

(1)

Amplification

122

(1)

Contrasting

122

(1)

Multiple labelling

122

(1)

References

123

(2)

In Vitro Methods for Testing Antigen-Antibody Reactions

125

(4)

Radioimmunoassay

126

(1)

Enzyme-linked immunosorbent assay (ELISA)

126

(1)

Western blotting

127

(1)

Dot blots

127

(1)

References

128

(1)

Applications of Immunocytochemistry

129

(12)

Histopathological diagnosis

129

(4)

Controls

130

(1)

Choice of antibody

131

(1)

Tips for diagnostic laboratories

131

(2)

Research

133

(1)

Quantification

133

(3)

Confocal microscopy

134

(1)

Flow cytometry and fluorescent antibody cell sorting (FACS)

135

(1)

Simpler methods of quantification

135

(1)

Non-immunocytochemical uses of labelled probes

136

(2)

Receptor localization

137

(1)

Lectin histochemistry

137

(1)

In situ hybridization of nucleic acids

138

(1)

References

138

(3)

Appendix: Technical Notes

141

(30)

Buffers for diluting antibodies and rinsing

141

(1)

Phosphate-buffered normal saline (PBS)

141

(1)

Tris-buffered normal saline (TBS)

141

(1)

Antibody diluent and storage of antibodies

142

(1)

Double dilutions

142

(1)

Adherence of preparations to slides

143

(1)

Coating slides with poly-L-lysine

143

(1)

Coating slides with silane

144

(1)

Blocking endogenous peroxidase reaction

144

(2)

Paraffin sections

144

(1)

Milder methods for cryostat sections and whole-cell preparations

145

(1)

Blocking endogenous biotin

146

(1)

Enzyme pre-treatment

146

(2)

Trypsin

146

(1)

Protease

147

(1)

Pepsin

147

(1)

Neuraminidase

148

(1)

Heat-mediated antigen retrieval using a microwave oven

148

(2)

Enzyme development methods

150

(6)

Peroxidase

150

(3)

Alkaline phosphatase

153

(1)

Glucose oxidase

154

(1)

β-D-Galactosidase

155

(1)

Intensifying the peroxidase/DAB reaction product

156

(2)

Following standard development

156

(1)

During development

156

(2)

Immunostaining methods

158

(3)

Initial procedures

158

(2)

Immunostaining - all preparations

160

(1)

Immunogold staining with silver enhancement

161

(3)

Silver acetate auto-metallography

163

(1)

Double immunoenzymatic staining

164

(2)

Primary antibodies from different species

164

(1)

Primary antibodies from the same species, heat-blocking method

165

(1)

Post-embedding electron microscopical immunocytochemistry using epoxy resin-embedded tissue and an indirect immunogold method

166

(2)

Absorption specificity control (liquid phase)

168

(1)

References

169

(2)

Index

171

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Introduction to Immunocytochemistry

Summary

Author Biography

Table of Contents

Supplemental Materials