Cytology, General Histology and Embryology - V. K. Napkhanyuk 2002

Methods of Histological Research
Post-vital Research Methods
Study of the Chemical Composition and Metabolic Characteristics of Cells and Tissues

Histochemical Methods

These methods make it possible to identify the localization of various chemical substances within the structures of Cells, Tissues, and Organs—including DNA, RNA, Proteins, CARBOHYDRATES, Lipids, Amino Acids, minerals, Vitamins, and enzyme activity. They are based on the Specificity of the reaction between a chemical reagent and a substrate found within cellular and tissue structures, as well as the coloration of the resulting reaction products.

Enzymatic controls are used to enhance reaction specificity.

When detecting Cytology/practical/13.html">Ribonucleic acid in cells, gallocyanin—a dye with basic properties—is used, and the presence of RNA is confirmed by a control Treatment with Ribonuclease, an enzyme that degrades RNA. Gallsocyanin stains RNA blue-purple. Following preliminary Treatment of the specimen with ribonuclease and subsequent staining with gallocyanin, no coloration is observed, thereby confirming the presence of RNA within the Structure.

Radioautography method

This method allows for the most comprehensive study of METABOLISM in various body structures. It is based on The Use of radioactive elements—phosphorus (³²P), carbon (¹⁴C), sulfur (³⁵S), hydrogen (³H)—and compounds labeled with them. Radioactive substances in histological sections are detected using a photographic emulsion applied to the specimen and subsequently developed. In areas of the specimen where the emulsion contacts a radioactive substance, a photoreaction occurs, resulting in The formation of exposed areas (tracks). This method makes it possible to determine the rate at which labeled Amino acids are incorporated into proteins, the synthesis of Nucleic Acids, and iodine metabolism in thyroid cells.

Interferometry

This method is used to determine the dry mass, concentration of dense substances, and total protein content in living and fixed cells.

Quantitative histochemical methods

These methods enable the Determination of the content of various substances in Cells and Tissues, as well as the localization of chemical components within specific cellular and tissue structures.

Cytospectrophotometry. A method for the quantitative study of intracellular substances based on their absorption spectra.

Cytospectrofluorometry. A method for the quantitative study of intracellular substances based on their fluorescence spectra.

Immunofluorescence assay methods

These are used to study Cell Differentiation processes and to detect specific chemical compounds and structures within cells. The foundation of these methods is the Antigen-Antibody Reaction. Every cell in the Organism possesses a specific antigenic profile, which is largely determined by its proteins. The reaction products can be stained and visualized using a luminescence Microscope.

Review Questions

1. Types of histological specimens.

2. MAIN STAGES OF preparing histological specimens for light Microscopy.

3. Main stages of preparing histological specimens for Electron microscopy.

4. Staining of specimens. Classification of histological Dyes.

5. The Essence of Tissue and organ fixation. Types of fixatives.

6. Embedding histological specimens in solid media.

7. Methods for in vivo and in vitro vital Study of Cells and tissues, and their characteristics.

8. Histochemical Research Methods.

9. Radioautography methods.

10. Immunofluorescence assay methods.

11. Quantitative histochemical research methods.

Sample Examination Questions

1. Vital research methods.

2. Post-Vital Research Methods.

3. Main stages of histological preparation manufacture.