Principles of Histochemistry
Histochemical analysis relies on a chemical reagent binding to a strictly defined substance within a cell or extracellular matrix. The resulting reaction product stands out visually from other structures because its color differs significantly from the original reagent, allowing researchers to accurately determine the biochemical composition and localization of a substance.
An exception to the rule of mandatory chemical reaction is provided by indifferent dyes, whose action is based on simple physical adsorption or physical dissolution of the dye within the tissue substrate.
Detection of Nucleic Acids
For the separate visualization of RNA and DNA, histology employs differentiated approaches that yield contrasting color patterns on slide preparations.
- RNA detection (Brachet's reaction). The specimen is treated with a mixture of methyl green and pyronin. RNA-containing structures (ribosomes, nucleoli) stain a rich magenta (crimson) color, while other components appear green. A classic example is pancreatic tissue: secretory cells actively synthesize protein, are rich in ribosomes, and thus show magenta cytoplasm and nucleoli. To control for specificity, the section is treated with the enzyme ribonuclease. It degrades RNA, and the magenta staining disappears, proving that RNA was indeed stained.
- DNA detection (Feulgen reaction). Periodic acid–Schiff base (Schiff reagent, or fuchsin-sulfurous acid) is used. In this case, the DNA-containing nuclear chromatin acquires a cherry or purple color. Nucleoli and cytoplasm are counterstained green. In liver cells, the distribution of staining is precisely the opposite of Brachet's reaction results.
Detection of Carbohydrates and Glycosaminoglycans
Polysaccharides (glycogen, glycoproteins) and mucus components are detected using the PAS reaction (Periodic acid–Schiff reaction). The method involves oxidation with periodic acid followed by the addition of Schiff reagent. PAS-positive structures acquire a purple or dark red color. For example, in a small intestine section, mucus in the cytoplasm of epithelial goblet cells stains bright purple.
To detect glycosaminoglycans (GAGs)—components of the amorphous extracellular matrix—the basic dye toluidine blue is used. It demonstrates metachromasia: the ability to shift its original blue color to red or purple upon binding to the tissue. The chemical basis of this phenomenon lies in the interaction of the dye with heteropolysaccharides rich in acidic groups (radicals). A clear example of metachromasia can be observed in the wall of the aorta.
Reactions for Proteins and Neutral Fats
To detect protein structures (amino acid groups), specific reagents are applied:
- Bromphenol blue: proteins stain dark purple.
- Ninhydrin-Schiff reagent: imparts a red hue to proteins.
Neutral fats and lipids are detected using Sudan III, a lipophilic indifferent dye. Sudan III does not undergo a chemical reaction; instead, it acts via physical dissolution within the tissue substrate. It penetrates the cell and dissolves in lipid droplets (similar to the action of osmium tetroxide), imparting a bright orange color to fat inclusions. An excellent example is a whole-mount spread of the greater omentum, where adipocytes with large orange fat droplets are clearly visible.