Introduction to immunocytochemistry / J.M. Polak, S. Van Noorden.

Format:

Book

Author/Creator:

Polak, Julia M.

Contributor:

Van Noorden, Susan.

Language:

English

Subjects (All):

Immunocytochemistry.

Immunohistochemistry.

Medical Subjects:

Immunohistochemistry.

Physical Description:

xv, 176 pages, 8 unnumbered pages of plates : illustrations (some color) ; 24 cm

Edition:

Third edition.

Other Title:

Immunocytochemistry

Place of Publication:

Oxford : BIOS Scientific Publishers, 2003.

Summary:

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.

Contents:

History and development 1

2. Production of Antibodies 5

Immunization 5

Testing 6

Region-specific antibodies 6

Monoclonal antibodies 8

Selection of antibodies by phage display 9

Characteristics of a 'good' antibody 10

3. Preparation of Tissue for Immunocytochemistry 13

Fixation 13

Cross-linking fixatives 14

Precipitant fixatives 15

Combination fixatives 15

Fixed, paraffin-embedded tissue 16

Fresh material

frozen sections and cell preparations 16

Frozen sections 17

Whole cell preparations 18

Pre-fixed, non-embedded tissue 20

Pre-fixed frozen sections 20

Pre-fixed Vibratome sections 21

Whole-mounts 21

Permeabilization 21

Freeze-dried tissue 22

Tissue storage 22

Paraffin blocks and sections 22

Frozen blocks and sections 23

Cell preparations 23

Adherence of sections and cell preparations to slides 24

Antigen retrieval in fixed tissues 24

Washing 24

Protease treatment 24

Heat-mediated antigen retrieval 26

Colour plate section 33

4. Visualizing the End-product of Reaction 45

Fluorescent labels 46

Advantages 46

Disadvantages 46

Uses of immunofluorescence 46

Fluorescein 47

Rhodamine 47

Phycoerythrin 48

AMCA 48

Other fluorophores 48

Fluorescent counterstains 48

Enzyme labels 49

Peroxidase 49

Alkaline phosphatase 52

Glucose oxidase 52

[beta]-d-Galactosidase 53

Gold labels 53

Colloidal gold 53

Nanogold 54

Other labels 55

Biotin 55

Haptens 55

Radioisotopes 55

5. Non-specific Staining due to Tissue Factors 59

Causes of non-specific binding 60

Charged sites 60

Hydrophobic attraction 60

Fc receptors 60

Prevention of non-specific binding 60

Other problems 61

Endogenous enzymes 61

Endogenous biotin 61

Autofluorescence 61

6. Methods 63

Buffers 63

Antibody diluent and storage 64

Antibody dilution relative to reaction time, temperature and technique 65

Nature of antibodies (IgG) 67

Application of antibodies to preparations 69

Direct (one-step) method 70

Indirect (two-step) method 72

Three-layer methods 73

Avidin-biotin methods 76

7. Testing Antibodies: Specificity and Essential Controls 81

Testing a new primary antibody 81

A primary antibody with a known localization 81

A primary antibody with an unknown localization 85

Negative control for polyclonal antibodies

normal serum 85

Negative controls for monoclonal antibodies 85

Testing for non-specific binding of second and third reagents 86

Non-specific or unwanted specific staining due to antibody factors 86

Unwanted specific staining of unknown antigens 86

Non-specific binding of antisera to basic proteins 86

Unwanted specific cross-reactivity of anti-immunoglobulins 87

Cross-reactivity of the primary antibody with related antigens 87

Remedies for non-specificity due to tissue factors 89

Blocking binding sites with normal serum 89

Absorption with tissue powder 89

Remedies for non-specificity due to heterogeneity of the antibody 89

Dilution 89

Affinity purification 89

Remedies for non-specificity due to cross-reactivity 90

Essential staining controls 90

Negative controls 90

Positive controls 90

Experimental controls 91

8. Increasing Sensitivity and Enhancing Standard Methods 93

Increasing sensitivity 93

Immunogold with silver enhancement 93

Build-up methods 94

Tyramine signal amplification (TSA) 97

Intensification of the peroxidase/DAB/H[subscript 2]O[subscript 2] product 100

Post-reaction intensification 100

Intensification during the peroxidase reaction 101

9. Multiple Immunostaining 103

Double direct immunostaining with separately labelled primary antibodies 103

Double immunostaining with primary antibodies raised in different species, or of different immunoglobulin sub-class 104

Double immunoenzymatic method 104

Double immunofluorescence method 106

Triple immunostaining 106

Unlabelled primary antibodies from the same species 106

The problem 106

Elution methods 107

Indirect double immunostaining without elution 109

10. Immunocytochemistry for the Transmission Electron Microscope 115

Principles 115

Fixation 115

Pre-embedding immunocytochemistry 116

Non-embedding immunocytochemistry 116

Processing to resin 117

Labels 118

Sectioning resin blocks 119

Pre-treatment 120

Immunolabelling procedure 121

Immunolabelling with peroxidase 121

Amplification 122

Contrasting 122

Multiple labelling 122

11. In Vitro Methods for Testing Antigen-Antibody Reactions 125

Radioimmunoassay 126

Enzyme-linked immunosorbent assay (ELISA) 126

Western blotting 127

Dot blots 127

12. Applications of Immunocytochemistry 129

Histopathological diagnosis 129

Controls 130

Choice of antibody 131

Tips for diagnostic laboratories 131

Research 133

Quantification 133

Confocal microscopy 134

Flow cytometry and fluorescent antibody cell sorting (FACS) 135

Simpler methods of quantification 135

Non-immunocytochemical uses of labelled probes 136

Receptor localization 137

Lectin histochemistry 137

In situ hybridization of nucleic acids 138

Buffers for diluting antibodies and rinsing 141

Phosphate-buffered normal saline (PBS) 141

Tris-buffered normal saline (TBS) 141

Antibody diluent and storage of antibodies 142

Double dilutions 142

Adherence of preparations to slides 143

Coating slides with poly-L-lysine 143

Coating slides with silane 144

Blocking endogenous peroxidase reaction 144

Paraffin sections 144

Milder methods for cryostat sections and whole-cell preparations 145

Blocking endogenous biotin 146

Enzyme pre-treatment 146

Trypsin 146

Protease 147

Pepsin 147

Neuraminidase 148

Heat-mediated antigen retrieval using a microwave oven 148

Enzyme development methods 150

Peroxidase 150

Alkaline phosphatase 153

Glucose oxidase 154

[beta]-D-Galactosidase 155

Intensifying the peroxidase/DAB reaction product 156

Following standard development 156

During development 156

Immunostaining methods 158

Initial procedures 158

Immunostaining

all preparations 160

Immunogold staining with silver enhancement 161

Silver acetate auto-metallography 163

Double immunoenzymatic staining 164

Primary antibodies from different species 164

Primary antibodies from the same species, heat-blocking method 165

Post-embedding electron microscopical immunocytochemistry using epoxy resin-embedded tissue and an indirect immunogold method 166

Absorption specificity control (liquid phase) 168.

Notes:

Previous ed.: 1997.

Includes bibliographical references and index.

ISBN:

1859962084

OCLC:

51194081