Immunocytochemistry

TABLE OF CONTENTS -- Chapter-1 Antibodies and Antisera -- I. General Aspects of Antibodies and Antigen-Antibody Interactions -- II. Antiserum /Antibody Specificity -- A. Methods for Examining the Antiserum /Antibody Specificity -- B. Antibodies as Site- or Region-Specific Reagents -- C. Methods for Examining the Specificity of Antiserum /Antibody Interaction with Tissue-Bound Antigen: Controls -- 1. Definitions of Specificity -- 2. First-Level Controls -- a. Causes of Falsely Positive Absorption Controls -- i. Presence of Antigen-Binding Complexes -- ii. Presence of Antibodies to Contaminating Antigens, Present in the Same Compartments as the Antigen of Interest -- iii. Overabsorption of Low-Avidity Antibodies -- iv. Destruction of Antigens by Antisera -- v. Paradoxical Enhancement of Staining Intensity after Antigen Absorptions -- b. Causes of Falsely Negative Absorption Controls -- i. Complement-Mediated Binding of Immunoglobulins to Tissue -- ii. Basic Peptides and Poly-L-Lysine Bind IgG by a Saturable Mechanism -- 3. Second-Level or Staining Controls -- a. Deletion of all Antibodies -- b. Deletion of the Primary Antibody -- D. Independent Methods for Establishing Immunocytochemical Specificity -- References -- Chapter-2 Fixation and Tissue Pretreatment -- I. Fixation -- A. Introduction -- B. Formaldehyde -- C. Glutaraldehyde -- D. Acrolein -- E. Carbodiimide, Imidates, and Benzoquinone (Parabenzoquinone, PBQ) -- F. DEPC -- G. Osmic Acid or Osmium Tetroxide -- H. Precipitating Fixatives -- I. Combination Fixatives -- J. General Aspects of Fixation -- II. Tissue Pretreatment -- A. Isolated Cells, Cultured Cells, Imprints, and Tissue Fragments -- B. Vibratome® Sectioning -- C. Cryostat Sectioning -- D. Paraffin and Alternative Light Microscopy Embedding Media -- E. Freeze-Drying and Freeze-Substitution -- F. Plastic Embedding Techniques -- 1. Epon®-Araldite® Plastics -- 2. Polar Resins -- a. Lowicryl® K4M -- b. LR White -- G. Additional Embedding Media -- H. Ultracry otomy -- I. Comparisons and Recommendations -- References -- Chapter-3 Immunocytochemical Detection Systems -- I. General Aspects — Direct vs. Indirect Techniques -- II. Labeled and Unlabeled Antibody Detection Techniques -- A. Immunofluorescence -- 1. Fluorochromes and Labeling Techniques -- 2. Microscopy and Associated Methods for Fluorescence Detection -- 3. Fading -- 4. Counterstains -- B. Peroxidase-Based (Labeled and Unlabeled) Methods -- 1. HRP Cytochemistry -- a. Precision -- b. Sensitivity and Detection Efficiency: Intensification Methods -- c. Alternative Chromogens -- d. Specificity: Blocking of Endogenous Peroxidase-Like Activities -- 2. Immunoperoxidase (Peroxidase-Labeled Antibody) Methods -- 3. Unlabeled Antibody Methods -- a. Immunoglobulin Bridge and PAP Methods -- b. Double PAP and Double Bridge Variants -- c. The Link Antibody (Link Reagent) -- d. PAP-Fab -- 4. Alternative Immunoenzyme Methods -- a. Alkaline Phosphatase -- b. Glucose Oxidase -- C. Protein A-Based Methods -- D. Avidin-Biotin Systems -- E. Colloidal Gold-Based Methods -- 1. General Aspects -- 2. Preparation of Colloidal Gold Particles -- 3. Conjugation of Proteins to Colloidal Gold Particles -- 4. Colloidal Gold Probes in Light Microscopy -- a. Light and Fluorescence Microscopic Detection -- b. Immunogold-Silver Methods -- 5. Applications at the Scanning and Transmission Electron Microscopic Level -- 6. Other Applications of Colloidal Gold -- F. Ferritin-Based Methods -- G. Other Particulate Markers -- H. Radioactive Labeling of Antibodies or Protein A -- I. Hybrid Antibodies, Chimeral Antibodies, and Haptenated Antibodies -- J. Additional Markers -- III. Labeled Antigen Detection Methods -- A. Conceptual Origins -- B. Radioimmunocytochemistry (RICH) -- C. Gold-Labeled Antigen Detection (GLAD) -- D. Monoclonal Gold-Labeled Antigen Detection (CLONO-GLAD) -- IV. Conclusions and Suggestions -- A. Light and Fluorescence Microscopy -- B. Postembedding Procedures for TEM -- C. Pre-Embedding Procedures for TEM -- D. SEM -- References -- Chapter-4 Section Pretreatment, Epitope Demasking, and Methods for Dealing with Unwanted Staining -- I. Section Attachment to Slides and Free-Floating Sections -- II. Removal of Embedding Media -- III. Pretreatment of Sections to Remove Endogenous Interfering Activities -- IV. Demasking of Epitopes -- V. Pretreatment, Dilutions, and Application of Primary Antibodies -- A. Purification of Antisera and Antibodies -- B. Dilutions, Time of Incubation, Sensitivity, and Detection Efficiency -- C. Prozone Phenomenon: Implications for Quantitation -- D. Temperature -- E. Storage of Antisera and Dilutions -- F. Application of Antisera to Sections -- VI. Buffers and Detergents -- A. Buffers -- B. Detergents -- VII. Special Considerations for Postembedding Immunoelectron Microscopy -- VIII. Approaches to Avoid Unspecific Staining -- References -- Chapter-5 Associated Methods -- I. Immunocytochemical Demonstration of Multiple Antigens -- A. Adjacent and Mirror Section Methods -- B. Elution Double (Multiple) Staining Methods and Sequential Staining Methods -- C. Nonelution Double and Multiple Staining Methods for Light Microscopy -- 1. Direct Immunocytochemical Methods -- 2. Methods Employing Primary Antisera from Different Species and Species-Specific Second Antibodies -- 3. Methods Employing Primary Antisera from the Same Species in Indirect Methods -- D. Double and Multiple Staining Techniques for Electron Microscopy -- 1. Direct Immunocytochemical Methods -- 2. GLAD Method -- 3. Protein A-Colloidal Gold Method -- 4. Two-Face Staining Method -- 5. Primary Antisera from Different Species and Gold-Conjugated Species-Specific Second Antibodies -- 6. Methods Based on Destruction of Antigen-Combining Sites on Second Antibodies -- 7. Combinations of Gold-Silver- and Peroxidase-Based Methods -- 8. Additional Combined Methods and Quantitative Implications -- 9. Immunogold-Silver Approaches -- II. Immunocytochemistry of Living Cells -- III. Combinations of Immunocytochemistry with Other Techniques -- A. Combinations with Histology -- B. Formaldehyde-Induced Fluorescence of Monoamines -- C. Autoradiographic Techniques -- D. Retrograde Axonal Transport and Microinjection Techniques -- IV. Receptor Studies -- V. Quantitation -- References -- Appendix -- I. Coupling of Peptide Haptens to Carrier Proteins -- A. Carbodiimide Coupling -- B. Glutaraldehyde Coupling -- II. Purification of Antisera -- A. Purification of IgG by Affinity Chromatography and Preparation of F(ab)2 Fragments -- B. Coupling of Proteins or Peptides to Cyanogen Bromide-Activated Sepharose® 4B -- C. Tissue Powder Absorption -- III. Cytochemical Models for Assaying Antibody Specificity and Method Sensitivity and for Screening for Monoclonal Antibodies -- A. Peptide Antigens -- B. Other Antigens -- IV. Fixatives -- A. Formaldehyde-Based Fixatives -- 1. 4% Paraformaldehyde in 0.1 M Sodium Phosphate Buffer, pH 7.3 -- 2. Formaldehyde Fixation at Optimized pH -- 3. Bouin's Fluid -- 4. Zamboni's (Stefanini's) Fixative -- 5. Periodate-Lysine-Paraformaldehyde (PLP) -- 6. Formol Sublimate -- B. Glutaraldehyde-Based Fixatives -- 1. The Kamovsky-Type Mixtures -- 2. Acrolein-Glutaraldehyde Combinations -- 3. Picric Acid-Paraformaldehyde-Glutaraldehyde (PPG) -- 4. Carbodiimide-Glutaraldehyde -- 5. Parabenzoquinone-Glutaraldehyde and Parabenzoquinone- Glutaraldehyde-Formaldehyde (PG and PGF) -- 6. Periodate-Lysine-Paraformaldehyde-Glutaraldehyde (PLPG) -- C. Acrolein -- D. Diimidoesters -- E. Osmic Acid (Osmium Tetroxide) -- F. Carbodiimide -- G. Parabenzoquinone (Benzoquinone, PBQ) -- H. Diethy lpyrocarbonate -- I. Camoy's Fixative and Modifications -- 1. Camoy's Fixative -- 2. Modified Camoy (MOCA) -- 3. Methacam and Modified Methacam -- J. The Saint-Marie Procedure -- V. Tissue Pretreatment and Embedding Procedures -- A. Pre-Embedding (Nonembedding) Procedures -- 1. Isolated Cells, Glands, Tissues, and Monolayer Cultures -- a. Dehydration-Rehydration Permeabilization -- b. Detergent-Based Permeabilization -- c. Controls -- 2. General Guidelines for Preembedding Staining of Cells Cultured on Plastic Surfaces -- 3. Pre-Embedding Staining of Cryostat or Vibratome® Sections and Slices -- a. Cryostat Sections -- b. Vibratome® Sections -- 4. Pre-Embedding Staining: General Considerations -- B. Postembedding Procedures for Electron Microscopy or Light Microscopy -- 1. Cultured or Isolated Cells -- 2. Tissue Specimens: Embedding in Nonpolar Resins -- 3. Tissue Specimens: Embedding in Lowicryl® K4M — Low- Temperature Procedure -- 4. Tissue Specimens: Embedding in Lowicryl® K4M — Rapid and Intermediate Procedures -- 5. Tissue Specimens: Ultracryotomy -- a. Fixation -- b. Washing and Cryoprotection -- c. Freezing -- d. Sectioning -- e. Immunocytochemical Staining -- f. Contrasting -- C. Postembedding Procedures Primarily for Light Microscopy -- 1. Standard Paraffin Embedding Methods -- 2. Cryostat Sectioning -- 3. Vibratome® Sectioning -- 4. Embedding in Polyethylene Glycol (PEG) -- 5. Freeze-Drying and Vapor-Fixation -- VI. Labeling of Antibodies -- A. Fluorochroming of Proteins -- B. Preparation of Colloidal Gold Conjugates -- 1. Preparation of Colloidal Gold Particles -- a. Citrate Method -- b. Ascorbate Method -- c. White Phosphorus Method -- d. Thiocyanate Gold -- e. Ultrasonicated Gold -- f. The Tannic Acid Method of Slot and Geuze -- g. Colloidal Silver -- 2. Practical Notes on the Preparation of Colloidal Gold and Silver -- 3. Coupling of Antibodies and Other Reporter Proteins to Colloidal Gold (Silver) -- a. Protein Concentration Isotherms -- b. Determining Optimal pH for Coupling -- c. The Coupling Procedure -- d. Testing Gold Conjugate -- VII. Postembedding and Nonembedding Staining for Light or Fluorescence Microscopy -- A. Pretreatment of Sections and Application of Primary Antiserum -- 1. Paraffin Sections -- 2. Epon® or Araldite® Sections -- 3. Pretreatment of Hydrated Sections -- 4. Application of Primary Antiserum -- 5. Rinsing of Sections -- B. Indirect Immunofluorescence -- C. PAP Procedure and Peroxidase Visualization and Intensification Methods -- D. Indirect Immunoperoxidase or Immunoalkaline Phosphatase Staining -- E. ABC Method -- F. APAAP Method -- G. Immunogold-Silver Staining -- 1. The Silver Nitrate Method -- 2. The Silver Lactate Method -- 3. Special Considerations -- H. Clono-GLAD Method -- I. Radioimmunocytochemistry (RICH) -- VIII. Postembedding Staining for Electron Microscopy -- A. Pretreatment of Grids and General Considerations -- B. Incubation with Primary Antibodies -- C. Peroxidase-Antiperoxidase (PAP) Staining -- D. Gold-Labeled Antigen Detection (GLAD) -- E. Immunogold Staining -- F. Protein A-Gold Staining -- IX. Methods for Enlarging Gold Particle Size by Silver Enhancement -- A. Ultrastructural Autometallography -- B. Silver Enhancement with Protecting Colloid -- C. Physical Development with L4 Emulsion as Silver Donor -- X. Multiple Staining Methods -- A. Antibody Elution Technique -- B. Formaldehyde Blocking Technique -- XI. Abbreviations -- References -- Index.