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 vii
viii Contents 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 6.1.1 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
Contents ix 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... 83 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 13.1 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
x Contents 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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