Since antibodies tagged with markers have been developed, immunocytochemistry has become the method of choice for identifying tissue substances or for the localisation of nucleic acid in tissue by in situ hybridisation in molecular biology. Resin-embedded tissue is routinely used and new techniques are constantly introduced. Thus, the novice entering these fields has a breathtaking variety of methods open to him. 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 and appropriate choices made.
In the first part of the book, the background of the various resins available are discussed and information on inexpensive alternative technologies where they exist is provided. The various steps involved in tissue processing, beginning with fixation, are first described in theory, then detailed protocols are presented for their application, including troubleshooting sections. The second part rationalises the great variety of labelling methods that are commonly used for "on-section" cytochemistry and immunocytochemistry, where colloidal gold is currently overwhelmingly the most popular marker. The principles behind the usage of various markers and their limitations and advantages are discussed.

Inhalt

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°C) Temperature Protocols.- 3.3.2 Protocol for Room Temperature Rapid Polymerisation.- Dehydration.- Infiltration.- Polymerisation.- 3.3.3 Protocol for Cold Temperature (O°C) Polymerisation.- Dehydration.- Infiltration.- Polymerisation.- 3.4 Protocols Employing Dehydration of Tissue at Cold 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 Pri…