Fine Structure Immunocytochemistry

1 Introduction to Immunocytochemistry and Historical Background.- 1.1 Approaches to Immunocytochemistry.- 1.2 Criteria for an Electron-Microscopic Immunocytochemical Technique.- 1.3 Problems with the Pre-Embedding Techniques.- References.- 2 Fine Structure Preservation.- 2.1 Introduction.- 2.2 Fine-Structure Evaluation in Practice.- 2.2.1 Fine-Structure Stabilization by Fixation.- 2.2.2 Contrasting.- 2.2.3 Analysis.- 2.2.4 Interpretation.- 2.2.5 Selective Destruction.- 2.3 Summary.- References.- 3 Fixation for Fine Structure Preservation and Immunocytochemistry.- 3.1 Fine Structure Preservation.- 3.1.1 Glutaraldehyde.- 3.1.2 Formaldehyde.- 3.1.3 Effects of Aldehydes on Cell Components Other than Proteins.- 3.1.4 Cross-Linking Agents Other than Glutaraldehyde and Formaldehyde.- 3.2 Factors Affecting the Quality of Fixation for Fine Structure Preservation.- 3.2.1 Concentration and Length of Fixation.- 3.2.2 Temperature of Fixation.- 3.2.3 pH and the Buffer Vehicle.- 3.2.4 Osmolarity and Ionic Strength of the Buffer Vehicle.- 3.2.5 Purity of the Aldehyde Fixative; Storage and Disposal.- 3.2.6 Tissue Type.- 3.2.7 Methods of Fixation.- 3.2.8 Rate of Fixative Penetration.- 3.3 Fixation Artefacts.- 3.4 Effect of Fixatives on Enzyme Activity.- 3.4.1 Effect of Glutaraldehyde on Enzyme Activity.- 3.4.2 Effect of Formaldehyde on Enzyme Activity.- 3.5 Fixation for Immunocytochemistry.- 3.5.1 Effect of Glutaraldehyde on Antigenicity.- 3.5.2 Effect of Formaldehyde on Antigenicity.- 3.5.3 Effect of Acrolein on Antigenicity.- 3.5.4 Effect of Osmium Tetroxide on Antigenicity.- 3.5.5 Effect of Factors Affecting Cross-Linking on Antigenicity.- 3.5.6 Assessing the Effects of Fixation on Antigenicity.- 3.6 A Concluding Remark.- References.- 4 Embedding Media for Section Immunocytochemistry.- 4.1 Resin-Free Sections - Temporary Embedement.- 4.1.1 The PEG Method.- 4.1.2 The Poly-Methylmethacrylate Method.- 4.2 Permanent Embedding Media.- 4.2.1 Water-Miscible Media.- 4.2.2 New Acrylic Resins.- 4.2.3 Epoxy Resins.- 4.3 Freeze Substitution.- 4.3.1 Rapid Freezing.- 4.3.2 Freeze Substitution and Immunolabelling.- References.- Chapters 5 Cryo and Replica Techniques for Immunolabelling.- 5.1 Cryo Sectioning Techniques.- 5.1.1 Historical Perspectives.- 5.1.2 Theory of Freezing.- 5.1.3 The Knife.- 5.1.4 The Microtome.- 5.1.5 Cryo Sectioning Theory.- 5.1.6 The Hydrated Cryo Sectioning Technique.- 5.1.7 Tokuyasu Thawed Cryo-Section Technique.- 5.2 Freeze-Fracture and Replica Labelling Methods.- 5.2.1 Fracture-Label Method.- 5.2.2 Label-Fracture Method.- 5.2.3 Fracture-Flip.- 5.3 Other Replica Techniques for Labelling.- 5.3.1 Surface Replica Methods.- 5.3.2 Replicas of Labelled Sections.- References.- 6 Elementary Immunology.- 6.1 Basic Immunology.- 6.1.1 The Immune Response.- 6.1.2 Cells of the Immune System.- 6.2 The Structure of Antibodies.- 6.3 The Biological Functions of Immunoglobulins.- 6.3.1 lgG.- 6.3.2 lgM.- 6.3.3 IgA.- 6.3.4 lgD and lgE.- 6.3.5 Generation of Antibody Diversity.- 6.4. The Nature of Antigenicity.- 6.4.1 Antigen Size - Carriers and Haptens.- 6.4.2 Antigen Presentation.- 6.4.3 Proteins as Antigens.- 6.4.4 The Interaction of Antibodies with Their Antigens.- 6.5 Practical Aspects of Immunology.- 6.5.1 Preparation of Antibodies.- 6.5.2 Production of Polyclonal Antibodies.- 6.5.3 Monoclonal Antibodies.- 6.5.4 Anti-Peptide Antibodies.- 6.5.5 Use of Molecular Biology Approaches to Express Immunoglobulin Molecules in Cells.- 6.5.6 Purification of Antibodies.- 6.6 Affinity Purification.- 6.7 Characterization of Antibodies.- 6.8 Precipitin Techniques.- 6.9 Immunoblotting.- 6.10 Immunoprecipitation.- 6.11 Immunoassay.- References.- 7 Labelling Reactions for Immunocytochemistry.- 7.1 Historical Perspectives.- 7.2 Labelling in Practice.- 7.2.1 Purity of Antibody: Use of Light Microscopy.- 7.2.2 Class of Antibody; Hybrid Antibodies.- 7.2.3 Antibody Concentrations.- 7.2.4 Time, Temperature and Repeated Applications of Antibody.- 7.2.5 Avoiding Non-Specific Labelling.- 7.2.6 Evaluation and Control of Labelling Reactions.- 7.2.7 The Immunocytochemical Controls.- 7.2.8 The Biological Controls.- 7.2.9 Problems in Labelling.- 7.2.10 Accessibility Problems on Sections.- 7.2.11 Sensitivity of Labelling.- 7.3 Approaches for Single Labelling.- 7.4 Approaches for Double Labelling.- 7.4.1 Double Direct Labelling.- 7.4.2 Double Indirect - Simultaneous.- 7.4.3 Double Indirect - Sequential.- 7.4.4 Multiple-Labelling Using Serial-Sections.- 7.4.5 Avidin-Biotin Systems.- 7.4.6 Resolution.- References.- 8 Particulate Markers for Immunoelectron Microscopy.- 8.1 Colloidal Gold.- 8.1.1 Introduction.- 8.1.2 Properties of Gold Colloids.- 8.1.2.1 Gold Particles in Microscopy.- 8.1.2.2 Electrolyte-Induced Aggregation.- 8.1.3 Preparation of Gold Colloids.- 8.1.3.1 Methods.- 8.1.3.2 Number and Concentration of Gold Particle/Complexes.- 8.1.4 Factors that Influence Protein-Gold Complex Formation.- 8.1.4.1 Protein Concentration.- 8.1.4.2 Surfaces of Protein and Gold.- 8.1.4.3 pH and Ionic Strength.- 8.1.5 Preparation of Protein Gold Complexes.- 8.1.5.1 Selection and Adjustment of Complexing Conditions.- 8.1.5.2 Complex Preparation, Purification and Storage.- 8.1.5.3 Specific Protein-Gold Complexes.- 8.1.6 Ferritin.- 8.1.6.1 Method for Conjugating Protein to Ferritin.- 8.1.7 Imposil.- Appendix. Silver Enhancement Procedure.- References.- 9 Non-Immunological High-Affinity Interactions Used for Labelling.- 9.1 Lectins.- 9.1.1 Simple Sugar Chemistry.- 9.1.2 Glycoproteins.- 9.1.3 Lecting Labelling.- 9.2 Protein A.- 9.2.1 Properties of Protein A.- 9.2.2 Species Specificity.- 9.2.3 Binding to Fab Regions of Antibodies.- 9.2.4 Kinetics of Reaction and the Effect of pH.- 9.3 Protein G.- 9.3.1 Use of Protein G for Immunocytochemistry.- 9.4 Avidin-Biotin Interactions.- 9.4.1 Properties of Egg-White Avidin.- 9.4.2 Streptavidin.- 9.4.3 Biotin.- 9.4.4 Biotinylation of Proteins.- 9.4.5 The Use of a Linker in the Biotinylation Reaction.- 9.4.6 Avidin-Biotin for Affinity Labelling Studies.- 9.4.7 Biotinylation Studies with Living Cells.- 9.4.8 Streptavidin-Gold.- 9.5 Miscellaneous Labelling Approaches Relying on Non-Immunological High Affinity Interactions.- 9.5.1 The Enzyme Gold Method.- 9.5.2 Receptor/Ligand-Gold Complexes.- 9.5.3 Toxin-Gold Conjugates.- 9.6 In-Situ Hybridization.- References.- 10 Preembedding Immuno-Labelling.- 10.1 Permeabilization.- 10.1.1 Chemistry of Detergents.- 10.1.2 Non-Ionic Detergents - Triton X-100.- 10.1.3 Saponin.- 10.1.4 Digitonin.- 10.1.5 Bacterial Toxins.- 10.1.6 Permeabilization for EM Labelling in Practice.- 10.1.7 The Saponin/Thick Section Approaches.- 10.1.8 The Freeze-Thaw Approach.- 10.2 Markers for Preembedding Labelling.- 10.2.1 Horseradish Peroxidase.- 10.2.2 Particulate Markers for Preembedding.- 10.3 Preembedding Studies of the Nucleus.- 10.4 Preembedding Labelling for Studies of the Nervous System.- 10.5 The Use of Dinitrophenol IgG Conjugates.- 10.6 Agarose Gel Method for Preembedding.- References.- 11 Quantitative Aspects of Immunocytochemistry.- 11.1 General Comments.- 11.2 Basic Stereology.- 11.2.1 General Introduction to Stereology.- 11.3 Estimation of Volume Density and Surface Density in Practice.- 11.3.1 Point and Intersection Counts.- 11.4 Estimation of Volume of Reference Space.- 11.4.1 Organ or Tissue.- 11.4.2 Estimating Mean Cell Volume.- 11.5 Stereological Sampling in Practice.- 11.5.1 Measurement of Lv in Practice.- 11.6 Quantitation in Immunocytochemistry.- 11.6.1 Use of Point and Intersection Counting for Relating the Number of Gold Particles to the Volume, Surface or Length of Structures in Sections.- 11.6.2 Labelling Efficiency.- 11.6.3 Estimation of LE in Practice.- 11.7 Approaches for Quantitation Ignoring Labelling Efficiency.- 11.8 Absolute Quantitation Making Assumptions About Labelling Efficiency.- 11.9 Negation of the Labelling Efficiency Factor: The Matrix Gel Method for Quantitating Souble Antigens Which Are Available in Pure Form.- 11.9.1 Possible Limitations of this Approach.- 11.10 Quantitative Model System Developed for Small Molecular Weight Neurotransmitters.- 11.11 Sensitivity of Labelling: Limits of Detection.- 11.11.1 Soluble Proteins.- 11.11.2 Membrane Proteins.- 11.12 Steric Hindrance.- 11.13 Amplification of Gold Labelling and Quantitation.- 11.14 Signal-to-Noise Ratio and the Concentration of Antigen: Those Few Gold Particles!.- References.- 12 An Overview of Techniques for Labelling at the EM Level.