Immunohistochemistry: Basics and Methods

Immunohistochemistry: Basics and Methods

by Igor B. Buchwalow, Werner Bïcker

Paperback(2010)

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Product Details

ISBN-13: 9783642425028
Publisher: Springer Berlin Heidelberg
Publication date: 10/30/2014
Edition description: 2010
Pages: 153
Product dimensions: 6.10(w) x 9.25(h) x 0.01(d)

Table of Contents

1 Antibodies for Immunohistochemistry 1

1.1 Structure of Antibodies 2

1.2 Polyclonal Antibodies 4

1.3 Mouse Monoclonal Antibodies 4

1.4 Rabbit Monoclonal Antibodies 5

1.5 Protein A and Protein G in Immunohistochemistry 7

References 8

2 Antibody Labeling and the Choice of the Label 9

2.1 Covalent Labeling of Antibodies 9

2.2 Non-Covalent Labeling of Primary Antibodies with Labeled Fab Fragments 10

2.3 Enzyme Labels for Light Microscopy 13

2.4 Fluorophore Labels for Fluorescence Microscopy 15

2.5 Colloidal Gold Labels for Electron Microscopy 16

References 17

3 Probes Processing in Immunohistochemistry 19

3.1 Fixation in Immunohistochemistry 19

3.1.1 Fixation in Alcohols and Acetone 20

3.1.2 Fixation in Formaldehyde 20

3.1.3 Effect of Formaldehyde Fixation on Antigen-Antibody Binding 21

3.2 Paraffin Sections for Immunohistochemical Analysis 22

3.2.1 Embedding and Cutting 22

3.2.2 Mounting Paraffin Sections onto Slides 23

3.3 Cryosections for Immunohistochemical Analysis 24

3.4 Buffers for Washing and Antibody Dilution 25

3.5 Mounting Following Immunohistochemical Staining 27

3.6 Storage Following Immunohistochemical Staining 28

References 28

4 Working with Antibodies 31

4.1 Direct Immunostaining Method 31

4.2 Indirect Immunostaining Method 33

4.3 The Choice of Antibodies 35

4.3.1 The Choice of Primary Antibodies 35

4.3.2 The Choice of Secondary Antibodies 36

4.4 Optimal Concentration of the Antibody 37

4.5 Specificity Controls in Immunohistochemistry 38

References 39

5 Background Staining, Autofluorescence and Blocking Steps 41

5.1 Fc Receptors 41

5.2 Endogenous Peroxidase 42

5.3 Endogenous Alkaline Phosphatase 43

5.4 Endogenous Biotin 43

5.5 Autofluorescence or "The Wood Through the Trees" 44

References 46

6 Immunostaining Enhancement 47

6.1 Antigen Retrieval 47

6.3.1 Heat-Induced Antigen Retrieval 48

6.1.2 Proteolytic Antigen Retrieval 50

6.2 Signal Amplification 51

6.2.1 Avidin-Biotin Complex 52

6.2.2 Chain Polymer-Conjugated Technology 53

6.2.3 Tyramide Signal Amplification 54

6.2.4 Amplification of the Amplifier 58

References 58

7 Multiple Multicolor Immunoenzyme Staining 61

7.1 Simultaneous Immunoenzymatic Double Staining 61

7.2 Sequential Immunoenzymatic Double/Multiple Staining 63

7.3 "Stripping" Buffers for Sequential Immunoenzymatic Double Staining 65

7.4 Nuclear Counterstaining Following Immunoenzyme Labeling 66

References 66

8 Multiple Immunofluorescence Staining 69

8.1 Double Immunofluorescence Indirect Staining Using Primary Antibodies Raised in Two Different Host Species 70

8.2 Using Primary Antibodies of Different IgG Isotype 71

8.3 Using Haptenylated Primary Antibodies 74

References 76

9 Antigen Detection on Tissues Using Primary Antibody Raised in the Same Species 77

9.1 Haptenylation of Primary Antibodies with the Following Use of Secondary Antibodies Recognizing the Corresponding Hapten 78

9.2 Blocking Endogenous Tissue Immunoglobulins Homologous to Primary Antibody by Preincubation with Unconjugated Fab Fragments 79

References 81

10 Probes for Staining Specific Cellular Organelles 83

10.1 Nuclear Markers 83

10.1.1 Nuclear Fluorescent Counterstaining 85

10.1.2 Cell-Proliferation Nuclear Markers 85

10.1.3 Nuclear Envelope Markers 85

10.1.4 Nucleolar Markers 86

10.2 Probes for Mitochondria 87

10.3 Probes for Endoplasmic Reticulum and Golgi Apparatus 88

10.4 Endocytic Pathways 89

10.5 Probes for Cytoskeleton 91

10.6 Phalloidin Probes for Actin Filaments 92

References 94

11 The Use of Epitope Tags in Histochemistry 95

References 98

12 Immunohistochemistry at the Ultrastructural Level 99

12.1 Colloidal Gold Conjugates 99

12.2 Fixation for Ultrastructural Immunohistochemistry 100

12.3 Resin Etching and Heat-Induced Antigen Unmasking in Resin Sections 101

12.4 Immunogold Labeling Procedure 104

References 107

13 Diagnostic Immunohistochemistry 109

3.1 Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast 110

13.1.1 Human Keratins and Their Expression Patterns in Breast Epithelium 110

13.1.2 Progenitor Cell Keratins K5/K14 110

13.1.3 K17: Keratin of Basal/Myoepithelial Cells 112

13.1.4 Glandular Keratins 113

13.1.5 K1/K10: Major Keratins of Keratinocyte Differentiation and Keratinization 114

13.1.6 Myoepithelial Differentiation Markers 115

13.1.7 Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast 117

13.1.8 Carcinomas of Luminal Phenotype (Luminal Type Carcinomas) in the Breast 117

13.1.9 Tumors of Basal Phenotype 118

13.1.10 Squamous Cell Carcinomas 119

13.2 Tissue Microarrays 122

References 125

14 A Picture Is Worth a Thousand Words 129

14.1 Brightfield Microscopy 129

14.1.1 How to Set Up a Light Microscope Properly for Transmitted Light Illumination: Köhler Illumination 130

14.1.2 The Choice of Microscope Objectives 131

14.2 Fluorescence Microscopy 134

14.2.1 The Choice of Filter Cubes for Fluorescence Microscopy 134

14.2.2 The Choice of Fluorophores for Multiple Immunostaining 137

References 139

Glossary 141

Index 151

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