Main description:

Since antibodies tagged with markers have been developed, immunocytochemistry has become an important method for identifying tissue substances and the localisation of nucleic acid in tissue by in situ hybridisation in molecular biology. This laboratory book covers the embedding of tissue using less sensitive epoxy resin methods to the more sensitive procedures employing the acrylics. The possibilities are discussed and results are presented so that an understanding of the techniques can be acquired.

Contents:

I: Resin Embedding.- 1 The Strategic Approach.- Overview.- Planning a Project.- 1.1 Fixation Strategies.- 1.1.1 Chemical Fixation.- Tissue Fixation.- Modes of Fixation.- 1.1.2 Cryoprocedures.- Cryoimmobilisation and Resin Embedding.- Cryosubstitution.- Freeze-Drying.- Cryoultramicrotomy for Immunocytochemistry.- 1.2 Dehydration Strategies.- Choice of Dehydrating Solvent.- 1.3 Polymerisation Strategies.- 2 The Resins.- 2.1 Epoxy Resins.- 2.1.1 Araldite.- 2.1.2 Epon.- 2.1.3 Spurr's.- 2.1.4 Durcupan.- 2.2 Acrylic Resins.- 2.2.1 LR White.- Historical Perspective.- Versatility of LR White.- 2.2.2 LR Gold.- 2.2.3 The Lowicryls.- Historical Perspective.- Versatility of the Lowicryls.- Lowicryls K4M and HM20.- Lowicryls K11M and HM23.- Rapid Embedding Procedures for Lowicryls.- 2.2.4 Unicryl.- Historical Perspective.- Versatility of Unicryl.- Unicryl or Lowicryl?.- 3 Resin Embedding Protocols for Chemically Fixed Tissue.- 3.1 Tissue Handling.- 3.1.1 Free-Living Cells (and Cell-Fractions).- 3.1.2 Monolayers.- 3.1.3 Solid Tissue.- 3.2 Protocols Employing Full Dehydration of Tissue at Room Temperature (RT).- 3.2.1 Fixation.- Aldehyde Blocking.- 3.2.2 Protocol for Epoxy Resins with Ethanol.- Dehydration.- Infiltration.- Polymerisation.- 3.2.3 Protocol for Epoxy Resins with Acetone.- Dehydration.- Infiltration.- Polymerisation.- 3.2.4 Protocol for Acrylic Resins: LR White.- Dehydration.- Infiltration.- Polymerisation.- 3.2.5 Protocol for Acrylic Reisns: Lowicryls K4M/HM20 and Unicryl.- Dehydration.- Infiltration.- Polymerisation.- 3.3 Protocols Employing Partial Dehydration of Tissue at Room Temperature (RT).- 3.3.1 Fixation.- Fixation for Room Temperature Protocols.- Fixation for Cold (0 DegreesC) Temperature Protocols.- 3.3.2 Protocol for Room Temperature Rapid Polymerisation.- Dehydration.- Infiltration.- Polymerisation.- 3.3.3 Protocol for Cold Temperature (O DegreesC) Polymerisation.- Dehydration.- Infiltration.- Polymerisation.- 3.4 Protocols Employing Dehydration of Tissue at Cold Temperatures (O DegreesC to ?20 DegreesC).- 3.4.1 Fixation.- 3.4.2 Protocol for Dehydration down to ?20 DegreesC.- Dehydration.- Infiltration.- Polymerisation.- 3.5 Protocols Employing Dehydration of Tissue at Progressively Lower Temperatures (PLT: ?35 DegreesC to ?50 DegreesC).- 3.5.1 Apparatus for PLT.- 3.5.2 Fixation.- 3.5.3 Protocol for PLT to ?35 DegreesC.- Dehydration.- Infiltration.- Polymerisation.- 3.5.4 Protocol for PLT to ?50 DegreesC.- 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 DegreesC to Room Temperature).- Vapour Fixation.- Resin Infiltration.- Polymerisation.- 4.4.4 Protocol for Acrylic Resins (?20 DegreesC to ?80 DegreesC).- 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 DegreesC): LR White.- Cold Temperatures (?20 DegreesC): LR White.- 5.2.2 Chemical Catalytic Polymerisation and the Lowicryls.- Room Temperature: Lowicryls.- Cold Temperature (O DegreesC): Lowicryls.- Cold Temperatures (?20 DegreesC): Lowicryls.- Low Temperatures (?35 DegreesC): Lowicryls.- Experimental Parameters for Chemical Catalytic Polymerisation: Lowicryls.- 5.2.3 Chemical Catalytic Polymerisation and Unicryl.- Room Temperature: Unicryl.- Cold Temperature (O DegreesC): Unicryl.- Cold Temperatures (?20 DegreesC): Unicryl.- Low Temperatures (?35 DegreesC): 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 DegreesC to ?20 DegreesC).- Low Temperatures (?35 DegreesC to ?50 DegreesC).- Very Low Temperatures (?50 DegreesC to ?80 DegreesC).- 5.4 'Uncatalysed' LR White.- 5.4.1 Heat Polymerisation Methods.- 5.4.2 Chemical Catalytic Polymerisation Methods.- Room Temperature.- Cold Temperature (O DegreesC).- Cold Temperature (?20 DegreesC).- 5.4.3 Ultraviolet Light Polymerisation Methods.- Room Temperature.- Cold Temperatures (O DegreesC to ?20 DegreesC).- 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.