Essence and principles of histochemistry
Histochemical analysis is based on the fact that a chemical reagent binds to a strictly defined substance within a cell or the extracellular matrix. The resulting reaction product stands out visually against the background of other structures because its color differs sharply from the hue of the initial reagent. Thus, the biochemical composition and localization of a substance can be reliably determined.
An exception to the rule of obligatory chemical reaction is indifferent dyes, whose action is based on simple physical adsorption or physical dissolution of the dye in the tissue substrate.
Methods for detecting nucleic acids
For the separate visualization of RNA and DNA, histology uses differentiated approaches that yield contrasting color patterns on preparations.
- Detection of RNA (Brachet reaction). The preparation is treated with a mixture of methyl green and pyronin. Structures containing RNA (ribosomes, nucleoli) are stained crimson by pyronin, while other components turn green. A classic example is pancreatic tissue: secretory cells actively synthesize protein and are rich in ribosomes, which is why their cytoplasm and nucleoli appear crimson. To control specificity, the section is treated with the enzyme ribonuclease. It destroys RNA, and the crimson staining disappears, proving that RNA was indeed what was stained.
- Detection of DNA (Feulgen reaction). Periodic acid–Schiff reagent (Schiff's reagent, or fuchsin-sulfurous acid) is used. In this case, nuclear chromatin containing DNA acquires a cherry or purple color. Nucleoli and cytoplasm are counterstained green. Using liver cells as an example, it is clearly visible that the distribution of staining is completely opposite to the results of the Brachet reaction.
Detection of carbohydrates and glycosaminoglycans
Polysaccharides (glycogen, glycoproteins) and mucus components are detected using the PAS reaction (PAS-reaction). The acronym stands for Periodic Acid–Schiff (oxidation with periodic acid followed by the addition of Schiff's reagent). PAS-positive structures acquire a purple or dark red color. For example, in a small intestine preparation, mucus in the cytoplasm of villous epithelial goblet cells stains bright purple.
To detect glycosaminoglycans (GAGs)—components of the amorphous ground substance of connective tissue—the basic dye toluidine blue is used. It demonstrates the phenomenon of metachromasia: the ability to change its original blue color to red or purple upon binding to tissue. The chemical basis of this phenomenon lies in the interaction of the dye with heteropolysaccharides containing a large number of acidic groups (radicals). A clear example of metachromasia can be observed in an aorta wall preparation.
Reactions for proteins and neutral fats
To detect protein structures (amino acid groups), specific reagents are used:
- With bromophenol blue: proteins stain dark purple.
- Ninhydrin–Schiff reagent: imparts a red hue to proteins.
Neutral fats and lipids are detected using the dye Sudan III. It belongs to the group of lipophilic indifferent dyes. Sudan III does not enter into a chemical reaction, but works through physical dissolution in the substrate. It penetrates the cell and dissolves in lipid droplets (similar to the action of osmic acid), imparting a bright orange color to fat inclusions. An excellent example is a whole-mount film preparation of the greater omentum (where a piece of tissue is simply stretched on a slide), on which fat cells—adipocytes—with large orange fat droplets are clearly visible.
Briefly: answers to topical questions
Which structures stain green in the Brachet reaction and why?
In the Brachet reaction, DNA-containing nuclear structures and other structures appear green, whereas RNA-containing structures stain crimson. The reagent is a mixture of methyl green and pyronin: ribosomes and nucleoli are stained crimson by pyronin; nuclei (DNA) are described in sources as green.
What other indifferent dyes, besides Sudan III, are used to detect lipids?
Besides Sudan III, sources list the following for detecting fats/lipids:
- Sudan IV—stains fats red.
- Sudan Black B—stains fats black.
For the Sudan group of dyes, the mechanism is specified: they are indifferent, lipophilic, do not enter into a chemical reaction, and physically dissolve in fat and lipid droplets.
Also listed as special agents for detecting fats are: Oil Red O and Scarlet R—red; osmic acid—black; Nile blue sulfate—fatty acids dark blue, neutral fats red.
Brachet—RNA—crimson (via pyronin). Feulgen—DNA—cherry (via Schiff's reagent).
Class and exam questions
What is the main difference between the histochemical method and conventional staining?
Histochemistry is based on a specific chemical reaction between a reagent and a specific cellular substance, rather than simple dye precipitation.
Why is ribonuclease used in the Brachet reaction?
Ribonuclease serves to control specificity: the enzyme destroys RNA, after which the color disappears. This proves that RNA was specifically what stained crimson.
What is metachromasia using the example of toluidine blue?
This is the ability of a dye to change its color from blue to purple or red upon binding to the acidic groups of glycosaminoglycans.
Does Sudan III enter into a chemical reaction with fats?
No, it is an indifferent lipophilic dye. It physically dissolves in fat droplets, imparting an orange color to them.
What else to study on this topic
- The mechanism of polysaccharide oxidation by periodic acid in the PAS reaction
- Properties and types of Schiff's reagent (fuchsin-sulfurous acid)
- Whole-mount film preparations: differences from microtome sections
- Localization of glycosaminoglycans in the vascular wall
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