Immunocytochemistry

Lars-Inge Larsson, D.M.Sc., is Professor of Histochemistry and Head of the Department of Molecular Cell Biology, State Serum Institute, Copenhagen, Denmark. Dr. Larsson received his D.M.Sc. in 1975 at the Department of Histology, University of Lund, Sweden. From 1976 to 1982 he held an appointment as Associate Professor at the Institute of Medical Biochemistry, University of Aarhus, Denmark and was appointed Professor of Histochemistry in 1982. Between 1982 and 1987 he directed the first MRC Unit in Denmark, jointly supported by the Danish MRC and Cancer Society. In 1988 he constructed and was appointed head of the new Department of Molecular Cell Biology at the State Serum Institute in Copenhagen. Dr. Larsson is the author of over 200 scientific articles and books. He received the Boehringer-Ingelheim award in 1982, the Mack-Forster award (European Society for Clinical Investigation) in 1986, and the Anders Jahre and Lundbeck awards in 1987. He is, according to a recent survey made by the Institute of Scientific Information (lSI), among the 250 most cited primary authors in all scientific disciplines. His main area of interest is concerned with studies of neurohormonal peptides and biogenic amines and with the development of histochemical and biochemical methods for the study of these substances. He is the author of a very large number of original articles and reviews on immunocytochemical methodology.

• 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.