Contents. 11 The Use of Epitope Tags in Histochemistry References... 98

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1 Contents 1 Antibodies for Immunohistochemistry Structure of Antibodies Polyclonal Antibodies Mouse Monoclonal Antibodies Rabbit Monoclonal Antibodies Protein A and Protein G in Immunohistochemistry... 7 References Antibody Labeling and the Choice of the Label Covalent Labeling of Antibodies Non-Covalent Labeling of Primary Antibodies with Labeled Fab Fragments Enzyme Labels for Light Microscopy Fluorophore Labels for Fluorescence Microscopy Colloidal Gold Labels for Electron Microscopy References Probes Processing in Immunohistochemistry Fixation in Immunohistochemistry Fixation in Alcohols and Acetone Fixation in Formaldehyde Effect of Formaldehyde Fixation on Antigen Antibody Binding Paraffin Sections for Immunohistochemical Analysis Embedding and Cutting Mounting Paraffin Sections onto Slides Cryosections for Immunohistochemical Analysis Buffers for Washing and Antibody Dilution Mounting Following Immunohistochemical Staining Storage Following Immunohistochemical Staining References vii

2 viii Contents 4 Working with Antibodies Direct Immunostaining Method Indirect Immunostaining Method The Choice of Antibodies The Choice of Primary Antibodies The Choice of Secondary Antibodies Optimal Concentration of the Antibody Specificity Controls in Immunohistochemistry References Background Staining, Autofluorescence and Blocking Steps Fc Receptors Endogenous Peroxidase Endogenous Alkaline Phosphatase Endogenous Biotin Autofluorescence or The Wood Through the Trees References Immunostaining Enhancement Antigen Retrieval Heat-Induced Antigen Retrieval Proteolytic Antigen Retrieval Signal Amplification Avidin Biotin Complex Chain Polymer-Conjugated Technology Tyramide Signal Amplification Amplification of the Amplifier References Multiple Multicolor Immunoenzyme Staining Simultaneous Immunoenzymatic Double Staining Sequential Immunoenzymatic Double/Multiple Staining Stripping Buffers for Sequential Immunoenzymatic Double Staining Nuclear Counterstaining Following Immunoenzyme Labeling References Multiple Immunofluorescence Staining Double Immunofluorescence Indirect Staining Using Primary Antibodies Raised in Two Different Host Species Using Primary Antibodies of Different IgG Isotype Using Haptenylated Primary Antibodies References

3 Contents ix 9 Antigen Detection on Tissues Using Primary Antibody Raised in the Same Species Haptenylation of Primary Antibodies with the Following Use of Secondary Antibodies Recognizing the Corresponding Hapten Blocking Endogenous Tissue Immunoglobulins Homologous to Primary Antibody by Preincubation with Unconjugated Fab Fragments References Probes for Staining Specific Cellular Organelles Nuclear Markers Nuclear Fluorescent Counterstaining Cell-Proliferation Nuclear Markers Nuclear Envelope Markers Nucleolar Markers Probes for Mitochondria Probes for Endoplasmic Reticulum and Golgi Apparatus Endocytic Pathways Probes for Cytoskeleton Phalloidin Probes for Actin Filaments References The Use of Epitope Tags in Histochemistry References Immunohistochemistry at the Ultrastructural Level Colloidal Gold Conjugates Fixation for Ultrastructural Immunohistochemistry Resin Etching and Heat-Induced Antigen Unmasking in Resin Sections Immunogold Labeling Procedure References Diagnostic Immunohistochemistry Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast Human Keratins and Their Expression Patterns in Breast Epithelium Progenitor Cell Keratins K5/K K17: Keratin of Basal/Myoepithelial Cells Glandular Keratins K1/K10: Major Keratins of Keratinocyte Differentiation and Keratinization Myoepithelial Differentiation Markers

4 x Contents Keratins and Myoepithelial Markers as Diagnostic Markers in Proliferative Breast Disease and in Tumors of Breast Carcinomas of Luminal Phenotype (Luminal Type Carcinomas) in the Breast Tumors of Basal Phenotype Squamous Cell Carcinomas Tissue Microarrays References A Picture Is Worth a Thousand Words Brightfield Microscopy How to Set Up a Light Microscope Properly for Transmitted Light Illumination: Köhler Illumination The Choice of Microscope Objectives Fluorescence Microscopy The Choice of Filter Cubes for Fluorescence Microscopy The Choice of Fluorophores for Multiple Immunostaining References Glossary Index