Immunohistochemistry: Basics and Methods (eBook)

Chapter 1: Antibodies for Immunohistochemistry 10
Structure of Antibodies 11
Polyclonal Antibodies 13
Mouse Monoclonal Antibodies 13
Rabbit Monoclonal Antibodies 14
Protein A and Protein G in Immunohistochemistry 16
References 17
Chapter 2: Antibody Labeling and the Choice of the Label 18
Covalent Labeling of Antibodies 18
Non-Covalent Labeling of Primary Antibodies with Labeled Fab Fragments 19
Enzyme Labels for Light Microscopy 22
Fluorophore Labels for Fluorescence Microscopy 24
Colloidal Gold Labels for Electron Microscopy 25
References 26
Chapter 3: Probes Processing in Immunohistochemistry 27
Fixation in Immunohistochemistry 27
Fixation in Alcohols and Acetone 28
Fixation in Formaldehyde 28
Effect of Formaldehyde Fixation on Antigen-Antibody Binding 29
Paraffin Sections for Immunohistochemical Analysis 30
Embedding and Cutting 30
Mounting Paraffin Sections onto Slides 31
Cryosections for Immunohistochemical Analysis 32
Buffers for Washing and Antibody Dilution 33
Mounting Following Immunohistochemical Staining 35
Storage Following Immunohistochemical Staining 36
References 36
Chapter 4: Working with Antibodies 38
Direct Immunostaining Method 38
Indirect Immunostaining Method 40
The Choice of Antibodies 42
The Choice of Primary Antibodies 42
The Choice of Secondary Antibodies 43
Optimal Concentration of the Antibody 44
Specificity Controls in Immunohistochemistry 45
References 46
5: Background Staining, Autofluorescence and Blocking Steps 47
Fc Receptors 47
Endogenous Peroxidase 48
Endogenous Alkaline Phosphatase 49
Endogenous Biotin 49
Autofluorescence or ``The Wood Through the Trees´´ 50
References 52
Chapter 6: Immunostaining Enhancement 53
Antigen Retrieval 53
Heat-Induced Antigen Retrieval 54
Proteolytic Antigen Retrieval 56
Signal Amplification 57
Avidin-Biotin Complex 58
Chain Polymer-Conjugated Technology 59
Tyramide Signal Amplification 60
Amplification of the Amplifier 64
References 64
Chapter 7: Multiple Multicolor Immunoenzyme Staining 66
Simultaneous Immunoenzymatic Double Staining 66
Sequential Immunoenzymatic Double/Multiple Staining 68
``Stripping´´ Buffers for Sequential Immunoenzymatic Double Staining 70
Nuclear Counterstaining Following Immunoenzyme Labeling 71
References 71
Chapter 8: Multiple Immunofluorescence Staining 73
Double Immunofluorescence Indirect Staining Using Primary Antibodies Raised in Two Different Host Species 74
Using Primary Antibodies of Different IgG Isotype 75
Using Haptenylated Primary Antibodies 78
References 80
Chapter 9: Antigen Detection on Tissues Using Primary Antibody Raised in the Same Species 81
Haptenylation of Primary Antibodies with the Following Use of Secondary Antibodies Recognizing the Corresponding Hapten 82
Blocking Endogenous Tissue Immunoglobulins Homologous to Primary Antibody by Preincubation with Unconjugated Fab Fragments 83
References 85
Chapter 10: Probes for Staining Specific Cellular Organelles 86
Nuclear Markers 86
Nuclear Fluorescent Counterstaining 86
Cell-Proliferation Nuclear Markers 88
Nuclear Envelope Markers 88
Nucleolar Markers 89
Probes for Mitochondria 90
Probes for Endoplasmic Reticulum and Golgi Apparatus 91
Endocytic Pathways 92
Probes for Cytoskeleton 94
Phalloidin Probes for Actin Filaments 95
References 97
Chapter 11: The Use of Epitope Tags in Histochemistry 98
References 101
Useful Websites 101
Chapter 12: Immunohistochemistry at the Ultrastructural Level 102
Colloidal Gold Conjugates 102
Fixation for Ultrastructural Immunohistochemistry 103
Resin Etching and Heat-Induced Antigen Unmasking in Resin Sections 104
Immunogold Labeling Procedure 107
References 110
Useful Websites 110
Chapter 13: Diagnostic Immunohistochemistry 111
Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast 112
Human Keratins and Their Expression Patterns in Breast Epithelium 112
Progenitor Cell Keratins K5/K14 112
K17: Keratin of Basal/Myoepithelial Cells 114
Glandular Keratins 115
K1/K10: Major Keratins of Keratinocyte Differentiation and Keratinization 116
Myoepithelial Differentiation Markers 117
Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast 119
Carcinomas of Luminal Phenotype (Luminal Type Carcinomas) in the Breast 119
Tumors of Basal Phenotype 120
Squamous Cell Carcinomas 121
Tissue Microarrays 124
References 127
Chapter 14: A Picture Is Worth a Thousand Words 130
Brightfield Microscopy 130
How to Set Up a Light Microscope Properly for Transmitted Light Illumination: Köhler Illumination 131
The Choice of Microscope Objectives 132
Fluorescence Microscopy 135
The Choice of Filter Cubes for Fluorescence Microscopy 135
The Choice of Fluorophores for Multiple Immunostaining 138
References 140
: Index 150