Theory and Strategy in Histochemistry : A Guide to the Selection and Understanding of Techniques

This book has been written to help scientists in biomedical research and routine service laboratories select and develop histochemical and cytochemical techniques. It does not give exhaustive methodological descriptions, but instead answers questions like "How can I apply histochemistry to my investigation?" "What are some typical problems?" "How should I interpret the results?"

1: General Considerations.- 1 The Scope of Histochemistry.- 1.1 Histochemieal and Histological Methods.- 1.2 The Histochemical Reaction.- 2 The Structural and Chemical Basis for Histochemistry.- 2.1 Chemical Composition of Cells and Tissues.- 2.2 Structure and Function of the Eukaryotic Cell.- 2.3 The Prokaryotic Cell.- 2.4 The Composition of the Extracellular Matrix.- 3 Reagents.- 3.1 Preparation of Reagents.- 3.2 Solvents.- 3.3 Dyes.- 3.4 Enzymes as Reagents.- 2: Review of Techniques According to Mechanism.- 4 General Theory for Tissue Staining.- 4.1 Tissue Sections.- 4.2 Dye.- 4.3 Solvent.- 4.4 Additives.- 4.5 Bonds Formed Between Dye and Tissue.- 5 Blocking and Deblocking Reactions.- 5.1 Classification of Blocking Reactions According to Mechanism.- 6 Staining of Macromolecules on the Basis of Charge.- 6.1 Tissue Groups that Bind Cationic Dyes.- 6.2 Demonstration of Proteins by Binding of Anionic Dyes.- 6.3 The Mechanism of Staining with Anionic and Cationic Dyes.- 7 Staining Involving Metal Complex Dyes.- 7.1 Complex Compounds.- 7.2 Metal-Haematein Complexes.- 7.3 Chromium-Gallocyanin.- 8 Staining Based on Reductants and Oxidants.- 8.1 Redox Reactions.- 8.2 Classification of Tissue Bound Reductants and Oxidants.- 8.3 Methods for the Demonstration of Reductants and Oxidants.- 9 Staining Involving Covalent Bonds.- 9.1 Alkene Groups.- 9.2 1,2-Glycol and l-Amino-2-Hydroxyl Groups.- 9.3 Thiol (Mercapto or Sulphhydryl) and Disulphide Groups.- 9.4 Aromatic Groups.- 9.5 Amino Groups.- 9.6 Guanidyl Groups.- 9.7 Aldehyde Groups.- 9.8 Acid Groups.- 9.9 Purine-N-C1-Deoxyribose Glycoside Bond.- 3: Tissue Processing.- 10 Tissue Processing I: Overview.- 11 Tissue Processing II: Freezing.- 11.1 Purpose of Freezing Tissue.- 11.2 The Mechanism of Freezing ..- 11.3 The Further Preparation Following Freezing.- 12 Tissue Processing III: Fixation, General Aspects.- 12.1 Definition.- 12.2 Classification of Fixatives.- 12.3 Requirements for the Fixative and the Fixation Process.- 13 Tissue Processing IV: Applied Fixation.- 13.1 Reactions of the Fixatives.- 13.2 Fixation of Proteins.- 13.3 Fixation of Nucleoproteins and Nucleic Acids.- 13.4 Fixation of Carbohydrates.- 13.5 Fixation of Lipids.- 13.6 Fixation of Enzymes.- 13.7 Fixation of Inorganic Components.- 13.8 Fixation of Pigments.- 14 Tissue Processing V: Embedding.- 14.1 Embedding of Tissue.- 14.2 Dehydration.- 14.3 Clearing.- 14.4 Embedding Media.- 14.5 Storage of Embedded Material.- 15 Tissue Processing VI: Hard Tissues.- 15.1 Specimen Types.- 15.2 Fixation.- 15.3 Selection of Tissue Blocks for Further Preparation.- 15.4 Decalcification.- 15.5 Further Preparation Following Decalcification.- 15.6 Staining of Decalcified Sections.- 15.7 Frozen Sections.- 15.8 Preparation of Non-Decalcified Material.- 15.9 Other Special Methods for Hard Tissue.- 16 Tissue Processing VII: Post Treatment.- 16.1 Introduction.- 16.2 Removal of Surplus of Reagent.- 16.3 Further Treatment of the Section.- 16.4 Mounting Media.- 4: The Staining of Chemical Entities.- 17 Metals and Metal Salts.- 17.1 Occurrence.- 17.2 Micro-Incineration.- 17.3 Electron Microscopical X-Ray Microanalysis.- 17.4 Processing of Tissue..- 17.5 Principles for the Histochemical Detection of Metals.- 17.6 Detection Limits.- 17.7 Individual Methods for the Detection of Inorganic Constituents.- 18 Pigments.- 18.1 Description of Pigment Groups and Individual Pigments.- 18.2 The Histochemical Properties of the Individual Pigments.- 18.3 Determination of an Unknown Pigment.- 18.4 Individual Reactions for Pigments.- 19 Lipids.- 19.1 A Survey of Histochemical Reactions for Lipids.- 19.2 Pretreatment.- 19.3 Differential Extraction of Lipids.- 19.4 Masked Lipids.- 19.5 Strategy for the Histochemical Investigation of Lipids.- 19.6 Outline of Lipid Methods.- 19.7 Myelin Methods.- 20 Nucleic Acids.- 20.1 Methods for the Demonstration of Nucleic Acids.- 20.2 Basophilia.- 20.3 Feulgen’s Nucleal Reaction.- 20.4 Application of Reactions for Nucleic Acids.- 20.5 The-Bromo-2'-Deoxyuridine Method 271.- 20.6 In Situ Hybridization.- 20.7 Polymerase Chain Reaction.- 20.8 Control Methods in Nucleic Acid Histochemistry.- 21 Proteins.- 21.1 Introduction.- 21.2 Fixation.- 21.3 Demonstration.- 21.4 Reactions for Protein Bound Amino Acids.- 21.5 Demonstration of Elastin.- 21.6 Demonstration of Collagen.- 21.7 Amyloid.- 22 Carbohydrates.- 22.1 Introduction.- 22.2 Demonstration.- 22.3 Blocking and Extraction.- 22.4 Lectins.- 5: Enzyme Histochemistry.- 23 Enzyme Histochemistry I. General Considerations.- 23.1 Biochemical Aspects.- 23.2 Histochemical Aspects.- 24 Enzyme Histochemistry II. Hydrolases.- 24.1 Principles of Hydrolase Demonstration.- 24.2 Pretreatment.- 24.3 Incubation.- 24.4 Controls in the Histochemical Investigation of Enzyme Activity.- 24.5 Quantitation.- 24.6 Demonstration of Selected Hydrolases.- 25 Enzyme Histochemistry III. Oxidoreductases.- 25.1 Principles of the Cytochemical Demonstration of Anaerobic Dehydrogenases.- 25.2 Principles of the Cytochemical Demonstration of Peroxidases.- 25.3 Principles of the Cytochemical Demonstration of Oxidases.- 25.4 Demonstration of Selected Oxidoreductases.- 6: Other Techniques.- 26 Immunohistochemistry.- 26.1 Definition.- 26.2 Labelling of Antibodies.- 26.3 Immunostaining Methods.- 26.4 The Choice and Evaluation of an Immunohistochemical Staining Technique.- 26.5 Immunohistochemical Controls.- 26.6 Tissue Processing.- 26.7 Quantitation.- 27 Ultrastructural Cytochemistry and Immunocytochemistry.- 27.1 Problems in Ultrastructural Cytochemistry.- 27.2 Some Major Reaction Types.- 27.3 Immunocytochemistry.- 28 Quantitation in Histochemistry.- 28.1 General Considerations.- 28.2 Absorption Photometry.- 28.3 Fluorimetry..- 28.4 Reflection Contrast Photometry.- 28.5 Interferometry.- 28.6 Spectral Analysis.- 28.7 Analysis of Staining Kinetics.- 28.8 Some Applications of Quantitative Histochemistry.- 29 Autoradiography.- 29.1 Physical Principles of Autoradiography.- 29.2 Application.- 29.3 Isotopes.- 29.4 Preconditions for Autoradiographic Experiments.- 29.5 Light Microscopic Autoradiography.- 29.6 Resolution.- 29.7 Artifacts.- 29.8 Quantitation.- 29.9 Radiation Safety.- 30 Fluorescence Microscopic Methods in Histochemistry.- 30.1 Autofluorescence.- 30.2 Induced Fluorescence.- 30.3 Direct Fluorochromy.- 30.4 Indirect Fluorochromy = Immunofluorescence.- 30.5 Enzymatically Provoked Fluorescence.- 7: An Introduction to Applied Histochemistry.- 31 Applied Histochemistry ― An Overview.- 31.1 Tissue Processing.- 31.2 General Oversight Stains.- 31.3 Demonstration of Ionized or Ionizable Groups.- 31.4 Demonstration of Microorganisms.- 31.5 Demonstration of Metals.- 31.6 Demonstration of Pigments.- 31.7 Demonstration of Lipids.- 31.8 Demonstration of Nucleic Acids.- 31.9 Demonstration of Proteins.- 31.10 Demonstration of Carbohydrates.- 31.11 Demonstration of Enzyme Activity.- 31.12 The Use of Autoradiography.- 31.13 The Use of Fluorescence Microscopy.- 32 Applied Immunohistochemistry.- 32.1 Introduction.- 32.2 Use and Interpretation of Immunohistochemistry in Diagnostic Pathology.- 32.3 Diagnostic Applications.- 32.4 Immunohistochemistry of Immunologic Disorders.- 32.5 Immunohistochemistry in the Diagnosis of Tumours.- 32.6 Immunohistochemical Identification of Microorganisms.- Appendix A: Standardization of Staining Methods.- A.I General Considerations.- A.2 Examples of Staining Methods.- Appendix B: Quantitative Methods in Microscopy.- B.I Definitions.- B.2 Observations.- B.3 Stereology.- B.4 Special Stereological Tools.- B.5 Simple “Counting” Procedures.- B.6 Manipulating Digital Images.- B.7 Applications of Stereology in Pathology.- References.- Index of Constituents.- Index of Dyes.- Index of Methods.