Resin Microscopy and On-Section Immunocytochemistry

Temperatures (O°C to ?20°C).- 3.4.1 Fixation.- 3.4.2 Protocol for Dehydration down to ?20°C.- Dehydration.- Infiltration.- Polymerisation.- 3.5 Protocols Employing Dehydration of Tissue at Progressively Lower Temperatures (PLT: ?35°C to ?50°C).- 3.5.1 Apparatus for PLT.- 3.5.2 Fixation.- 3.5.3 Protocol for PLT to ?35°C.- Dehydration.- Infiltration.- Polymerisation.- 3.5.4 Protocol for PLT to ?50°C.- Dehydration.- Infiltration.- Polymerisation.- 4 Cryotechniques.- 4.1 Tissue Preparation for Freezing.- 4.2 Rapid Freezing and Apparatus Requirements.- 4.2.1 Plunge-Freezing.- 4.2.2 Propane Jet Freezing.- 4.2.3 Spray Freezing.- 4.2.4 Slam or Impact Freezing.- 4.2.5 High-Pressure Freezing.- 4.2.6 Source of Apparatus.- 4.2.7 Safety.- 4.3 Cryosubstitution.- 4.3.1 The Substitution Medium.- 4.3.2 The Temperature and Duration of Substitution.- 4.3.3 Apparatus for Substitution.- 4.3.4 Protocol for Epoxy Resins.- Substitution Medium.- Substitution.- Infiltration.- Polymerisation.- 4.3.5 Protocol for Acrylic Resins: LR White and Unicryl.- Substitution Medium.- Substitution.- Infiltration.- Polymerisation.- 4.3.6 Protocol for Acrylic Resins: Lowicryls K4M/HM20 and Unicryl.- Substitution Medium.- Substitution.- Infiltration.- Polymerisation.- 4.3.7 Protocol for Acrylic Resins: Lowicryls K11M/HM23.- Substitution Medium.- Substitution.- Infiltration.- Polymerisation.- 4.4 Freeze-Drying.- 4.4.1 Protocol for Epoxy Resins.- Vapour Fixation.- Resin Infiltration.- Polymerisation.- 4.4.2 Acrylic Resins for Freeze-Drying.- 4.4.3 Protocol for Acrylic Resins (O°C to Room Temperature).- Vapour Fixation.- Resin Infiltration.- Polymerisation.- 4.4.4 Protocol for Acrylic Resins (?20°C to ?80°C).- Vapour Fixation.- Resin Infiltration.- Polymerisation.- 4.5 Cryoultramicrotomy.- 4.5.1 Fixation.- 4.5.2 Freezing of Tissue.- 4.5.3 Sectioning and Storage of Grids.- 5 Methods for Resin Polymerisation.- 5.1 Heat Polymerisation Methods.- 5.1.1 Epoxy Resins III.- 5.1.2 Acrylic Resins: LR White.- 5.1.3 Acrylic Resins: Lowicryls.- 5.1.4 Acrylic Resins: Unicryl.- 5.2 Chemical Catalytic Polymerisation Methods.- 5.2.1 Chemical Catalytic Polymerisation and LR White.- Room Temperature: LR White.- Cold Temperature (O°C): LR White.- Cold Temperatures (?20°C): LR White.- 5.2.2 Chemical Catalytic Polymerisation and the Lowicryls.- Room Temperature: Lowicryls.- Cold Temperature (O°C): Lowicryls.- Cold Temperatures (?20°C): Lowicryls.- Low Temperatures (?35°C): Lowicryls.- Experimental Parameters for Chemical Catalytic Polymerisation: Lowicryls.- 5.2.3 Chemical Catalytic Polymerisation and Unicryl.- Room Temperature: Unicryl.- Cold Temperature (O°C): Unicryl.- Cold Temperatures (?20°C): Unicryl.- Low Temperatures (?35°C): Unicryl.- 5.3 Ultraviolet Light Polymerisation Methods.- 5.3.1 The Setting-up of Apparatus.- 5.3.2 LR Resins, Lowicryls and Unicryl.- Room Temperature.- Cold Temperatures (O°C to ?20°C).- Low Temperatures (?35°C to ?50°C).- Very Low Temperatures (?50°C to ?80°C).- 5.4 'Uncatalysed' LR White.- 5.4.1 Heat Polymerisation Methods.- 5.4.2 Chemical Catalytic Polymerisation Methods.- Room Temperature.- Cold Temperature (O°C).- Cold Temperature (?20°C).- 5.4.3 Ultraviolet Light Polymerisation Methods.- Room Temperature.- Cold Temperatures (O°C to ?20°C).- 6 Handling Resin Blocks.- 6.1 Sectioning Blocks.- 6.1.1 Epoxy Resins.- 6.1.2 Acrylic Resins: LR White (LR Gold).- 6.1.3 Acrylic Resins: Lowicryls.- 6.1.4 Acrylic Resins: Unicryl.- 6.2 Storing Blocks.- 6.2.1 Epoxy Resins.- 6.2.2 Acrylic Resins: LR White (LR Gold).- 6.2.3 Acrylic Resins: Lowicryls.- 6.2.4 Acrylic Resins: Unicryl.- II: On-Section Immunolabelling.- 7 Strategies in Immunolabelling.- 7.1 Colloidal Gold Strategies.- 7.1.1 Direct Methods.- 7.1.2 Indirect Methods.- Immunogold Staining (IGS) or Labelling.- Protein A-Gold.- Protein G-Gold.- Protein AG-Gold.- 7.1.3 Hapten- (and Haptenoid-)Based Methods.- Biotin.- Dinitrophenyl (DNP).- 7.2 Peroxidase Strategies.- 7.2.1 Direct Methods.- 7.2.2 Indirect Methods.- 7.2.3 The Peroxidase Antiperoxidase (PAP) Method.- 7.2.4 Hapten- (and Haptenoid-) Based Methods.- Biotin.- Dinitrophenyl (DNP).- 7.3 EM Double Immunolabelling.- 8 General Considerations.- 8.1 Resin Section Pretreatment.- 8.1.1. Etching (Epoxy Resin Sections only).- Semithin Sections.- Thin Sections.- 8.1.2 Trypsinisation.- 8.1.3 Inhibition of Endogenous Peroxidase (Immunoperoxidase only).- 8.1.4 Abolition of Aldehyde Groups.- 8.1.5 Osmium Removal.- 8.1.6 Uranium.- 8.1.7 Equilibration.- 8.2 Resin Section Immunolabelling.- 8.2.1 Specific Blocking.- 8.2.2 The Primary Reagent.- 8.2.3 Washing.- 8.2.4 The Secondary Detection System.- 8.2.5 DAB (Immunoperoxidase only).- 8.2.6 Photochemical Visualisation of Colloidal Gold and DAB (Silver Intensification).- Silver Development.- Silver Intensification of Colloidal Gold.- Silver Intensification of Diaminobenzidin e (DAB).- 8.2.7 Counterstaining.- Semithin Sections.- Thin Sections.- 8.2.8 Dehydration and Coverslipping of Semithin Sections.- 8.2.9 Air-Drying of Thin Sections.- 8.3 Controls in Immunocytochemistry.- 8.3.1 Reagent Controls.- Omitting the Primary Reagent.- Dilution Profiles.- Inappropriate Primary Antiserum.- Pre-Absorption of the Primary Antiserum.- 8.3.2 Tissue Controls.- 9 Immunolabelling Protocols for Resin Sections.- 9.1 Pretreatment Protocols.- 9.1.1 Protocol for Semithin Sections.- Epoxy Resin Section Pretreatment.- Epoxy and Acrylic Resin Section Pretreatment.- 9.1.2 Protocol for Thin Sections.- Epoxy Resin Section Pretreatment.- Epoxy and Acrylic Resin Section Pretreatment.- 9.2 Immunocolloidal Gold Labelling Protocols.- 9.2.1 Protocol for Direct Imrnunocolloidal Gold Labelling.- Incubation.- Visualisation for Semithin Sections.- Visualisation for Thin Sections.- 9.2.2 Protocol for Indirect Immunocolloidal Gold Labelling (IGS, Protein AlG).- Incubation.- 9.2.3 Protocol for Hapten-Based Immunocolloidal Gold Labelling (Biotin!Antibiotin or Avidin, DNP AntiDNP).- (i) Direct Methods.- (ii) Indirect Methods.- 9.3 Immunoperoxidase Labelling Protocols.- 9.3.1 Resin Section Pretreatment.- Inhibition of Endogenous Peroxidase.- 9.3.2 Protocol for Direct Immunoperoxidase Labelling.- Incubation.- Visualisation for Semithin Sections.- Visualization for Thin Sections.- 9.3.3 Protocol for Indirect Immunoperoxidase Labelling.- Incubation.- 9.3.4 Protocol for Hapten-Based Immunoperoxidase Labelling.- (i) Direct.- (ii) Indirect.- 9.3.5 Protocol for DNP Hapten Sandwich Staining Technique (DHSS).- 9.3.6 Protocol for 4-Layer DNP Hapten Sandwich Staining Technique (4-DHSS).- 9.4 EM Double Immunolabelling Protocols.- 9.4.1 Protocol for Immunocolloidal Gold Labelling.- (i) Direct Methods.- (ii) Indirect Methods.- 9.4.2 Protocol for Hapten-Based Methods.- (i) Direct.- (ii) Indirect.- 9.4.3 Protocol for Immunocolloidal Gold/Immunoperoxidase DAB Combined.- Incubation and Visualisation.- 9.5 Immunocolloidal Gold Labelling Protocols for Cryoultramicrotomy.- 9.5.1 Section Pretreatment.- 9.5.2 Abolition of Aldehyde Groups.- 9.5.3 Equilibration.- 9.5.4 Protocol for Direct Immunocolloidal Gold Labelling.- Incubation.- 9.5.5 Protocol for Indirect Immunocolloidal Gold Labelling (IGS, Protein A/G).- Incubation.- 9.5.6 Visualisation.- 10 Resin Embedding and Immunolabelling.- 10.1 Extraction by the Resin.- 10.2 Resin Cross-Linking.- 10.3 Penetration of Label.- 10.4 Surface Relief of Sections.- 10.5 Gold Preparations.- 10.6 Quantitation.- 10.7 Conclusion.- Appendix I Examples of Typical Resin Embedding Regimes for Immunocytochemistry.- I.1 Solid Tissue, Pellets and Agar Blocks - Immersion Fixation.- I.2 Solid Tissue - Light Perfusion Fixation.- I.3 Solid Tissue - Very Light Perfusion Fixation.- I.4 Cryosubstitution.- I.5 Project Planner.- Appendix II List of Suppliers.- EM General.- EM Apparatus.- Electron Microscopes.- Flow Cytometry.- References.