Histochemistry

Histochemistry: An Explanatory Outline of Histochemistry and Biophysical Staining describes the histochemical staining of cells and tissues as a major tool applied in biological and medical investigations, both in basic research and in practical applications such as clinical diagnosis. The book may be considered as a guide to understanding the scientific basis of staining procedures and alternate actions to take when common methods do not proceed as expected. The first chapter gives general theoretical ideas from which most part of the book is largely organized around. As the book considers the arts and crafts making up the practice of histochemistry and biological staining, emphasis is given to the common physicochemical aspects of the technically diverse methodologies involved. Hence, the author has drawn ideas and information from physicochemically and biochemically related fields, such as chromatography, pharmacology, photography, tanning, and textile dyeing. The bulk of the book is structured around groups of practical procedures, such as fixation, staining with dyestuffs, metal impregnation, and selective extraction as a test of significance. Lastly, general essays on the usefulness of staining theories and on the problems arising from reagent impurities are given as examples. The text is suitable for students and researchers in the fields of physiology, biochemistry, and pharmacology. Clinical laboratory scientists and those involved in the life sciences and biotechnology will benefit from reading the book.

1 An Overview of Biological Staining

1.1 What Information Does Biological Staining Provide?

1.2 How Do We Carry out Biological Staining? And Why Do We Do What We Do?

1.3 What is Present to Be Stained? Fixation and Its Alternatives

1.4 Why Does Anything Become Stained? An Account of Reagent-Tissue Affinities, and of Other Causes

1.5 Why Doesn't Everything Always Stain?

1.6 Why Does the Tissue Remain Stained after Its Removal from the Staining Solution?

1.7 How Do We Judge the Significance of Staining? Or Indeed, of Failure to Stain?

2 The Preparation of Material to Be Stained

3 Fixation and Other Loss-Limiting Procedures

3.1 Why We Need to Limit Losses: The Practical Options

3.2 Limitation of Losses without Chemical Modification of the Tissue

3.3 Fixatives: Substances Used to Chemically Modify Native Tissues

3.4 How is the Fixation of Tissues Achieved?

3.5 The Fixation of Substances Other Than Protein

3.6 Effects of Fixation on Biological Staining: Limitation of Losses of Tissue Materials

3.7 Effects of Fixation on Biological Staining: Increases In Staining and Changes in Staining Patterns Due to Chemical Modifications of the Tissues

3.8 Effects of Fixation on Biological Staining Due to Physical Modifications of the Tissues

3.9 Rates of Fixative Penetration

4 Staining Methods Involving Dyeing

4.1 What are Dyes and What is Dyeing? Definitions, Names and Descriptive Terms

4.2 Why are Dyes Colored? The Color, Dichroism, Fluorescence and Metachromasia of Dyes

4.3 Why Do Dyes Stain Anything? The Affinity Concept and Some Thermodynamic Formalism

4.4 Various Affinity Models for Biological Staining

Model 1: Solvent Non-Aqueous, Dye Non-Ionic, No Dye-Tissue Forces

Model 2: Solvent Non-Aqueous, Dye Non-Ionic, But Dye-Tissue Forces Occur: Van Der Waals Attractions, Hydrogen Bonding, Mordanting

Model 3: Aqueous Solvent, Dye and/or Tissue Non-Ionic, No Appreciable Dye-Tissue Attractions: Hydrophobic Bonding

Model 4: Dye and Substrate Ionic, All Other Variables Taken as Trivial

Model 5: Aqueous Solvent, Dye and Substrate Ionic, Dye-Dye Attractions Occur

4.5 Dye-Tissue Affinities in Numbers: Various Quantitative Measures

4.6 Sources of Affinity - A Practical Summary

4.7 Why Don't Dyes Stain Everything?

4.8 Preliminaries Concerning Substrate Density and the Color of Staining

4.9 A Dye May Prefer One Substrate to Another: Some Affinity Models

4.10 Some Substrates Stain or Destain Faster than Others

4.11 What are the Causes of Differential Staining Rates?

4.12 How is the Rate of Staining Affected by the Dye Used?

4.13 How Do the Various Tissue Substrates Influence Staining Rates?

4.14 How Does Substrate Geometry Influence Staining Rate?

4.15 How Do Solvent and Co-Solute Affect Staining Rates?

4.16 How Do Pre-staining Treatments of the Tissue Affect Staining Rates?

4.17 A Summary of the Causes of Differential Rates of Staining

4.18 Various Examples of Rate-Controlled Selective Staining Procedures

4.19 Rate-Controlled Selective Staining Involving "Colorless Dyes"

4.20 Everything May Stain, and Stain at the Same Rate - But in a Variety of Colors

5 Reactive Staining Methods

5.1 Some Definitions, Generalizations and Cautions

5.2 The Influence of Reagent Uptake on the Selectivity of Staining

5.3 The Influence of Reactivity on Reagent Selectivity

6 Staining Methods Involving Uptake of Metals

6.1 A General Model for Staining Methods Involving Uptake of Metal

6.2 How Does the Uptake of Metal Occur?

6.3 How is the Metal Visualized after Uptake?

6.4 Why Should Such Methods Display Selectivity?

6.5 Why are Many Metal Impregnation Methods So Fickle?

7 Enzyme Histochemistry

7.1 Essential Problems and Characteristics of Enzyme Histochemistry

7.2 Ultrastructural Localization of Enzymes versus Preservation of Their Biochemical Integrity

7.3 Substrates and Incubation Media

7.4 Formation and Properties of the Initial Reaction Product

7.5 Characteristics of the Capture-Visualization Steps

7.6 Properties of the Final Reaction Product

7.7 How Do We Interpret the Results of a Staining Procedure?

7.8 Peculiarities of Electron-Microscopic Enzyme Histochemistry

8 Immuno-Chemical Staining Using Labeled Antibody and Related Methods

8.1 Some Strategies of Staining

8.2 A Sketch of the Chemistry of Antibodies and Antigens, and of Their Interactions

8.3 Preparation of Antibodies and of Analogous Reagents

8.4 Nature and Preparation of the Label

8.5 Labeling Methods: Preparation of Labeled Antibodies and Related Materials

8.6 Nature of Visualizing Procedures

8.7 Specificity of the Staining

8.8 Some Factors Limiting Sensitivity

8.9 The Conflicting Problems of Preserving Antigen and Antigenicity (Or Antibody and Immunogenicity) and Cell Morphology

9 Some Miscellaneous Biological Staining Methods

9.1 An Introduction

9.2 Dyeing with Reagents That are Not Dyes

9.3 A Trapping Method: The Gram Reaction

9.4 Vital Staining Procedures

10 Peculiarities of Staining Plastic Sections

10.1 The State of the Art of Staining Plastic Sections

10.2 What is the Effect of the Fixative?

10.3 Some Properties and Chemistry of Plastic Embedding Media, and of the Relevant Monomers

10.4 Penetration of Reagents into Plastic Sections: Some Observations and a Rationale

10.5 A General Approach to Staining Plastic Sections, with Some Applications and Complications

10.6 Some Practical Implications of the General Model

11 The Interpretation and Analysis of Histochemical Observations

11.1 Checking the Meaning of Staining: An Introduction

11.2 The Use of Selective Extraction

11.3 The Use of Histochemical Blocking

11.4 The Use of Other Control Procedures

11.5 Does Staining Intensity Reflect Substrate Concentration?

11.6 Is the Stain Where the Substrate Was? The Localization Problem

11.7 Checking the Meaning of No-Staining: An Introduction

11.8 Have There Been Substrate Losses?

11.9 Is There Masking of Substrate?

11.10 Was There Staining, Which Has Been Lost?

11.11 The Inevitability, Value and Nature of Staining Theories

11.12 Uses and Abuses of Test Objects, Model Systems and Histochemical-Chemical Comparisons as Aids to Understanding Staining Results

11.13 The Baleful Influence of Reagent Impurities on the Interpretation of Staining Results

11.14 The Uses of Structure-Staining Correlations, Practical and Theoretical

11.15 A Summary of Some Practical Strategies

12 Supplements

12.1 Nomenclature of Traditional Color Theory: Chromagen, Chromophore and Auxochrome

12.2 Π Bonds and Σ Bonds: A Glance at the Valency of Carbon

12.3 Relation of Affinity to Other Thermodynamic Quantities

12.4 The Nature and Characteristics of Van Der Waals Attractions

12.5 The Hydrogen Bond

12.6 The Coordinative Bond

12.7 Hydrophobic Bonding

12.8 Quantitative Estimation of Affinity: Use of Freundlich and Langmuir Isotherms

12.9 Methods of Determining Half Staining Times and Activation Energies for the Staining of Tissue Sections

12.10 Aggregation of Dyes in Solution: Some Physical Chemistry

12.11 The Precipitation of Sparingly Soluble ("Insoluble") Salts

12.12 Some Structural Characteristics of Immunoglobulins

12.13 Photofading: Some Physico-Chemical Background to the Fading of Stained Preparations Following Exposure to Visible or Ultra Violet Radiation

References

Subject Index