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Life Sciences - Cell Biology | Resin Microscopy and On-Section Immunocytochemistry

Resin Microscopy and On-Section Immunocytochemistry

Newman, Geoffrey R., Hobot, Jan A.

1993, XIX, 221p. 25 illus..

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  • About this book

For the first time, fixation, resin embedding and immunocytochemistry, the subjects of innumerable publications, have been organised into a comprehensive and coherent scheme showing how the various methodologies interrelate. Commercially available resins and their mode of use are described, and the book considers the advantages for cytochemistry and immunocytochemistry of matching tissue fixation to processing and resin embedding. On-section single and double immunolabelling methods are covered, using immunocolloidal gold and immunoperoxidase.

Content Level » Professional/practitioner

Keywords » Colloidal Gold - Harz-Einbettung - Immunomicroscopy - Immunomikroskopie - Kolloidales Gold - Resin Embedding - microscopy - tissue

Related subjects » Biochemistry & Biophysics - Cell Biology - Immunology - Internal Medicine

Table of contents 

I: Resin Embedding.- 1 The Strategic Approach.- 1.1 Introduction.- 1.2 Overview.- 1.3 Strategies for Planning a Project.- 1.3.1 Introduction.- 1.3.2 Fixation Strategies.- 1.3.2.1 Chemical Fixation.- 1.3.2.2 Cryoprocedures.- 1.3.3 Dehydration Strategies.- 1.3.3.1 Choice of Dehydrating Solvent.- 1.3.4 Polymerisation Strategies.- 2 The Resins.- 2.1 Epoxy Resins.- 2.1.1 Introduction.- 2.1.2 Choice of Resin.- 22 Acrylic Resins.- 2.2.1 LR White.- 2.2.1.1 Introduction.- 2.2.1.2 Historical Perspective.- 2.2.1.3 Versatility of LR White.- 2.2.2 LR Gold.- 2.2.3 The Lowicryls.- 2.2.3.1 Introduction.- 2.2.3.2 Historical Perspective.- 2.2.3.3 Versatility of the Lowicryls.- 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.- 3.2.1.1 Aldehyde Blocking.- 3.2.2 Protocol 1: for Epoxy Resins.- 3.2.2.1 Dehydration.- 3.2.2.2 Infiltration.- 3.2.2.3 Polymerisation.- 3.2.3 Protocol 2: for Acrylic Resins.- 3.2.3.1 Dehydration.- 3.2.3.2 Infiltration.- 3.2.3.3 Polymerisation.- 3.3 Protocols Employing Partial Dehydration of Tissue at Room Temperature (RT).- 3.3.1 Fixation.- 3.3.1.1 Fixation for Room Temperature Protocols.- 3.3.1.2 Fixation for Cold (0°C) Temperature Protocols.- 3.3.2 Protocol 1: Room Temperature Rapid Polymerisation.- 3.3.2.1 Dehydration.- 3.3.2.2 Infiltration.- 3.3.2.3 Polymerisation.- 3.3.3 Protocol 2: Cold (0°C) Polymerisation.- 3.3.3.1 Dehydration.- 3.3.3.2 Infiltration.- 3.3.3.3 Polymerisation.- 3.4 Protocols Employing Dehydration of Tissue at Cold Temperatures (0°C to -20°C).- 3.4.1 Fixation.- 3.4.2 Protocol for Dehydration down to -20°C.- 3.4.2.1 Dehydration.- 3.4.2.2 Infiltration.- 3.4.2.3 Polymerisation.- 3.5 Protocols Employing Dehydration of Tissue at Progressively Lower Temperatures (PLT: -35°C to -50°C).62.- 3.5.1 Apparatus for PLT.- 3.5.2 Fixation.- 3.5.3 Protocol 1: PLT to -35°C.- 3.5.3.1 Dehydration.- 3.5.3.2 Infiltration.- 3.5.3.3 Polymerisation.- 3.5.4 Protocol 2: PLT to -50°C.- 3.5.4.1 Dehydration.- 3.5.4.2 Infiltration.- 3.5.4.3 Polymerisation.- 4 Cryotechniques.- 4.1 Tissue Preparation for Freezing.- 4.2 Rapid Freezing.- 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.- 43 Cryosubstitution.- 4.3.1 The Substitution Medium.- 4.3.2 The Temperature and Duration of Substitution.- 4.3.3 Apparatus for Substitution.- 4.4 Protocols for Cryosubstitution (Epoxy Resins).- 4.4.1 Protocol 1: Epoxy Resins.- 4.4.1.1 Substitution Medium.- 4.4.1.2 Substitution.- 4.4.1.3 Infiltration.- 4.4.1.4 Polymerisation.- 4.5 Protocols for Cryosubstitution (Acrylic Resins).- 4.5.1 Protocol 2: LR White.- 4.5.1.1 Substitution Medium.- 4.5.1.2 Substitution.- 4.5.1.3 Infiltration.- 4.5.1.4 Polymerisation.- 4.5.2 Protocol 3: Lowicryls K4M and HM20.- 4.5.2.1 Substitution Medium.- 4.5.2.2 Substitution.- 4.5.2.3 Infiltration.- 4.5.2.4 Polymerisation.- 4.5.3 Protocol 4: Lowicryls K11M and HM23.- 4.5.3.1 Substitution Medium.- 4.5.3.2 Substitution.- 4.5.3.3 Infiltration.- 4.5.3.4 Polymerisation.- 4.6 Freeze-Drying.- 4.7 Protocols for Freeze-Drying (Epoxy Resins).- 4.7.1 Protocol 1.- 4.7.1.1 Vapour Fixation.- 4.7.1.2 Resin Infiltration.- 4.7.1.3 Polymerisation.- 4.8 Protocols for Freeze-Drying (Acrylic Resins).- 4.8.1 Protocol 2:0°C to Room Temperature.- 4.8.1.1 Vapour Fixation.- 4.8.1.2 Resin Infiltration.- 4.8.1.3 Polymerisation.- 4.8.2 Protocol 3: -20°C to -80°C.- 4.8.2.1 Vapour Fixation.- 4.8.2.2 Resin Infiltration.- 4.8.2.3 Polymerisation.- 4.9 Cryoultramicrotomy.- 4.9.1 Fixation.- 4.9.2 Apparatus Requirements.- 4.9.3 Sectioning and Storage of Grids.- 5 Methods for Resin Polymerisation.- 5.1 Introduction.- 5.2 Heat Polymerisation Methods.- 5.2.1 Epoxy Resins.- 5.2.2 Acrylic Resins.- 5.2.2.1 LR White.- 5.2.2.2 Lowicryls.- 5.3 Chemical Catalytic Polymerisation Methods.- 5.3.1 LR White.- 5.3.1.1 Room Temperature.- 5.3.1.2 Cold Temperature (0°C).- 5.3.1.3 Cold Temperatures (-20°C).- 5.3.2 Lowicryls.- 5.3.2.1 Room Temperature.- 5.3.2.2 Cold Temperature (0°C).- 5.3.2.3 Cold Temperatures (-20°C).- 5.3.2.4 Low Temperatures (-35°C) 9.- 5.3.2.5 Experimental Parameters for Chemical Catalytic Polymerisation.- 5.4 Ultraviolet Light Polymerisation Methods.- 5.4.1 The Setting-up of Apparatus.- 5.4.2 LR Resins and Lowicryls.- 5.4.2.1 Room Temperature.- 5.4.2.2 Cold Temperatures (0°C to -20°C).- 5.4.2.3 Low Temperatures (-35°C to -50°C).- 5.4.2.4 Very Low Temperatures (-50°C to -80°C).- 5.5 ‘Uncatalysed’ LR White.- 5.5.1 Heat Polymerisation Methods.- 5.5.2 Chemical Catalytic Polymerisation Methods.- 5.5.2.1 Room Temperature.- 5.5.2.2 Cold Temperature (0°C).- 5.5.2.3 Cold Temperature (-20°C).- 5.5.3 Ultraviolet Light Polymerisation Methods.- 5.5.3.1 Room Temperature.- 5.5.3.2 Cold Temperatures (0°C to -20°C).- 6 Handling Resin Blocks.- 6.1 Sectioning Blocks.- 6.1.1 Epoxy Resins.- 6.1.2 Acrylic Resins.- 6.1.2.1 LR White (LR Gold).- 6.1.2.2 Lowicryls.- 6.2 Storing Blocks.- 6.2.1 Epoxy Resins.- 6.2.2 Acrylic Resins.- 6.2.2.1 LR White (LR Gold).- 6.2.2.2 Lowicryls.- II: On-Section Immunolabelling.- 7 Strategies in Immunolabelling.- 7.1 Introduction.- 7.2 Colloidal Gold Strategies.- 7.2.1 Direct Methods.- 7.2.2 Indirect Methods.- 7.2.2.1 Immunogold Staining (IGS) or Labelling.- 7.2.2.2 Protein A-Gold.- 7.2.2.3 Protein G-Gold.- 7.2.2.4 Protein AG-Gold.- 7.2.3 Hapten- (and Haptenoid-) Based Methods.- 7.2.3.1 Biotin.- 7.2.3.2 Dinitrophenyl (DNP).- 7.2.4 Silver Intensification.- 7.3 Peroxidase Strategies.- 7.3.1 Direct Methods.- 7.3.2 Indirect Methods.- 7.3.3 The Peroxidase Antiperoxidase (PAP) Method.- 7.3.4 Hapten- (and Haptenoid-) Based Methods.- 7.3.4.1 Biotin.- 7.3.4.2 Dinitrophenyl (DNP).- 7.3.5 Silver Intensification.- 7.4 EM Double Immunolabelling.- 8 General Considerations.- 8.1 Resin Section Pretreatment.- 8.1.1 Etching (Epoxy Resin Sections only).- 8.1.1.1 Semithin Sections.- 8.1.1.2 Thin Sections.- 8.1.2 Trypsinisation.- 8.1.3 Inhibition of Endogenous Peroxidase (Immunopeioxidase 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).- 8.2.7 Counterstaining.- 8.2.7.1 Semithin Sections.- 8.2.7.2 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 Introduction.- 8.3.2 Reagent Controls.- 8.3.2.1 Omitting the Primary Reagent.- 8.3.2.2 Dilution Profiles.- 8.3.2.3 Inappropriate Primary Antiserum.- 8.3.2.4 Pre-Absorption of the Primary Antiserum.- 8.3.3 Tissue Controls.- 9 Immunolabelling Protocols for Resin Sections.- 9.1 Pretreatment Protocols.- 9.1.1 Semithin Sections.- 9.1.1.1 Epoxy Resin Section Pretreatment.- 9.1.1.2 Epoxy and Acrylic Resin Section Pretreatment.- 9.1.2 Thin Sections.- 9.1.2.1 Epoxy Resin Section Pretreatment.- 9.1.2.2 Epoxy and Acrylic Resin Section Pretreatment.- 9.2 Immunocolloidal Gold Labelling Protocols.- 9.2.1 Visualisation.- 9.2.2 Direct Immunocolloidal Gold Labelling.- 9.2.2.1 Incubation.- 9.2.2.2 Visualisation.- 9.2.3 Indirect Immunocolloidal Gold Labelling (IGS, Protein A/G).- 9.2.3.1 Incubation.- 9.2.4 Hapten-Based Immunocolloidal Gold Labelling.- 9.2.4.1 DirectMethods.- 9.2.4.2 Indirect Methods.- 9.3 Immunoperoxidase Labelling Protocols.- 9.3.1 Resin Section Pretreatment.- 9.3.1.1 Inhibition of Endogenous Peroxidase.- 9.3.2 Visualisation.- 9.3.3 Direct Immunoperoxidase Labelling.- 9.3.3.1 Incubation.- 9.3.3.2 Visualisation.- 9.3.4 Indirect Immunoperoxidase Labelling.- 9.3.4.1 Incubation.- 9.3.5 Hapten-Based Immunoperoxidase Labelling.- 9.3.5.1 Direct.- 9.3.5.2 Indirect.- 9.3.5.3 DNP Hapten Sandwich Staining Technique (DHSS).- 9.3.5.4 4-Layer DNP Hapten Sandwich Staining Technique (4-DHSS).- 9.4 EM Double Immunolabelling Protocols.- 9.4.1 Immunocolloidal Gold Labelling.- 9.4.1.1 Visualisation.- 9.4.1.2 DirectMethods.- 9.4.1.3 Indirect Methods.- 9.4.1.4 Hapten-Based Methods.- 9.4.2 Immunocolloidal Gold/Immunoperoxidase DAB Combined..- 9.4.2.1 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 Direct Immunocolloidal Gold Labelling.- 9.5.4.1 Incubation.- 9.5.5 Indirect Immunocolloidal Gold Labelling (IGS, Protein A/G).- 9.5.5.1 Incubation.- 9.5.6 Visualisation.- 10 Resin Embedding and Immunolabelling.- 10.1 Penetration of Label.- 10.2 Resin Cross-Linking.- 10.3 Surface Relief of Sections.- 10.4 Quantitation.- 10.5 Conclusion.- Appendix I.- Examples of Typical Resin Embedding Regimes for Immunocytochemistry.- Appendix II.- List of Suppliers.- References.

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