Autoradiography and Immunocytochemistry

Author's PrefaceAcknowledgmentsChapter 1. Introduction 1.1 The Demand for Autoradiography and Immunocytochemistry 1.2 The Most Advantageous Use of the TechniquesChapter 2. Immunocytochemistry at the EM Level: Preparation of Labelled Antibodies 2.1 Production and Purification of Antibodies 2.1.1 Selection of Antigens 2.1.2 Purified Specific Antibodies 2.1.3 Partial Isolation of Immunoglobulins 2.1.4 Isolation of Immunoglobulins by Affinity Chromatography Using Protein A 2.2 Ferritin-Labelled Antibodies 2.2.1 Purification of Commercial Ferritin 2.2.2 Preparation of Ferritin-Immunoglobulin Conjugates 2.2.3 Proof of Conjugation 2.3 Purification of Ferritin-Antibody Conjugates 2.3.1 Methods for Purifying Ferritin-Antibody Conjugates 2.3.2 Assessment of the Purity of Conjugates 2.3.3 Dual-Labelling with Fluorescein and Ferritin 2.4 Enzyme-Labelled Antibodies 2.4.1 Preparation of Enzyme-Labelled Antibodies 2.4.2 Proof of Conjugation of Antibodies to Peroxidase 2.5 Heavy Atom Labelling of Antibodies 2.6 Immuno-Electron Microscopy Using Non-Conjugated Electron-Dense Markers 2.6.1 Hybrid Antibodies 2.6.2 Conditions of Storage of Enzyme-Antibody Conjugates, PAP Complex and Hybrid Antibodies 2.7 Lectins as Staining Reagents for Immuno-Electron Microscopy 2.7.1 Preparation of Ferritin-Conjugated LectinsChapter 3. Immunocytochemistry at the EM Level: Staining Antigens with Electron-Dense Reagents 3.1 Fixation of Tissues, Cells and Cell Fragments 3.2 General Remarks on 'Staining' with Antibodies 3.2.1 Direct and Indirect Staining 3.3 Staining with Ferritin-Labelled Antibodies (Fer-Ab) 3.3.1 Surface or Extracellular Antigens 3.3.2 Staining Sub-Cellular Fractions 3.3.3 Staining Cells After Digitonin Treatment 3.3.4 Use of Fixed and Frozen Sections 3.3.5 Applying Fer-Ab Conjugates to Ultrathin Sections 3.4 Controls in Immunoferritin Staining 3.5 Staining with Antibody Dually-Labelled with Fluorescein and Ferritin 3.6 Enhancement of the Size and Electron Opacity of Ferritin by Heavy Metal Staining 3.7 Methods of Applying Enzyme-Labelled Antibodies 3.7.1 Staining Cell Surfaces or Extracellular Antigens 3.7.2 Staining Sub-Cellular Fractions with Enzyme-Labelled Antibodies 3.7.3 Fixation, Freezing and Staining of Intracellular Antigens in Solid Tissues 3.7.4 Staining Thick Sections of Tissue Embedded in Polyethylene Glycol 3.7.5 Staining Ultrathin Resin Sections with Enzyme-Labelled Antibodies 3.7.6 Staining Ultrathin Frozen Sections with Enzyme-Labelled Antibodies 3.8 Controls when Staining with Enzyme-Labelled Antibodies 3.9 Multistep Antibody Staining Methods with Unmodified Proteins 3.10 The Use of Lectins in Demonstrating Antigenic Determinants or Other Relevant Saccharides 3.10.1 Staining Cell Surface Saccharide Residues Using Unconjugated Concanavalin A 3.10.2 Control Observations in Staining Experiments Using Lectins 3.11 Quantitative Studies Using Immuno-Electron Microscopy 3.11.1 Pattern Analyssi in EM Immunocytochemisytr 3.11.2 Resolution in EM ImmunocytochemisytrChapter 4. Preparation of Electron Microscope Autoradiographs 4.1 Radioisotopes in EM Autoradiography 4.1.1 The Likely Utility of Particular Radioisotopes for EM Autoradiography 4.2 Radiochemicals 4.2.1 Storage and Handling of Radiochemicals 4.2.2 Checking the Purity of Radiochemicals 4.3 Route of Administration of the Radiochemical 4.4 Dose of Radiochemical 4.4.1 Labelling of Tissues in Whole Animals 4.4.2 Labelling Tissue or Cells in Vitro 4.4.3 Determination of the Suitability of a Radiochemical Dose by LM autoradiography 4.5 Toxicity of Radioisotopes 4.6 Preparation of Radioactively-Labelled Tissues and Cells 4.6.1 Fixation, Dehydration and Embedding of Tissues 4.6.2 Preparation of LM Autoradiographs from Thick Resin Sections 4.6.3 Ultramicrotomy 4.7 Choice of Nuclear Emulsion 4.8 Methods of Applying Nuclear Emulsions 4.8.1 Darkroom Conditions 4.8.2 The Loop Technique 4.8.3 Flat Substrate Methods 4.9 Exposure 4.10 Development 4.10.1 Choice of Developer 4.10.2 Method of Developing 4.10.3 Standard Commercial Chemical Developers for Use with Ilford L4 and Kodak NTE 4.10.4 Fine Grain Development 4.10.5 The pH of Developers 4.11 Recovery of Specimens from their Supports 4.11.1 Grids on Corks 4.11.2 Specimens on Collodion-Coated Slides 4.11.3 Specimens on Formvar-Carbon-Coatde Slides 4.12 Post-Developmental Staining and Removal of Gelatin 4.12.1 Staining without Gelatin Removal 4.12.2 Staining After Removal of Gelatin 4.13 Preparation of Micrographs from EM Autoradiographs 4.14 Control Observations 4.14.1 Determination and Significance of Background Count Levels 4.14.2 Positive Chemography 4.14.3 Negative Chemography 4.14.4 Diffusion of Labelled Products 4.14.5 The Detection of Fixative-Mediated Tissue Binding of Precursors and Other Low Molecular Weight Substances 4.15 Efficiency in EM Autoradiography 4.16 Resolution in EM Autoradiographs 4.17 Applications of EM Autoradiography 4.17.1 The Localization of Diffusible Substances by EM Autoradiography 4.17.2 EM Autoradiographs of a Particle-Emitting IsotopesAppendix 1. Chemicals, Reagents and Methods Mostly to do with ImmunocytochemistryAppendix 2. Materials and Methods for AutoradiographyAppendix 3. List of SuppliersSubject Index