Understanding Tissue Staining: Methods and Mechanisms

Posted on Mar 14, 2025 in Chemistry

Tissue Staining: Methods and Mechanisms

Matching Dyes to Colors: According to their coloring:

• Topographic: Provides an overall view of a fabric’s structure (e.g., H&E).
• Cytological: Allows for intimate study of cells (e.g., Pap smears, panoptic cell structure).
• Histochemical: Tries to highlight chemical substances or a determined chemical function (e.g., Perls, PAS highlight tissue structures, MSB Masson (collagenase), Orcein (TC), silver impregnation (reticulin)).

According to the coloring procedure:

• Direct: Dyes in tissue simply by the affinity of both (e.g., eosin).
• Indirect: Requires the action of an enhancer or mordant.
  • Mordant: A substance that serves as a link between the tissue and the coloring agent (e.g., chromic potassium permanganate).
  • A substance that causes increased selectivity or intensity of staining without acting as a link (e.g., phenol).
  • When treating tissue with mordant and dye simultaneously in a single solution, the coloration is called lacquer (an indirect, one-step process).
  • When the dye and mordant enhancer act separately, it’s called two-stage indirect coloring.
• Progressive: The coloration is stopped when the desired color is obtained.
• Regressive: The tissue is overstained, then excess dye is removed through differentiation (e.g., ethanol, acetone).
• Impregnation: A method based on the deposit of precipitated heavy metal salts on or within cells and tissue constituents.

Staining Mechanisms

• Physical: Properties are bound by solution (the dye is more soluble in tissue than in the solvent) and impregnation (by molecular size or their large volume, selective precipitation remains trapped between fibers and cells).
• Chemical: A chemical reaction between the dye and the resulting structure creates a new color.
• Physical-Chemical: Occurs via electrostatic attractions between the dye and the tissue.

Matching Dyes to Tissue Response:

• Natural: Scarce and derived from certain plants and insects (e.g., carmine, hematoxylin, safranin).
• Artificial: Very numerous derivatives of aniline.
  • According to their chromophore group:
  • Nitrated or nitro (-NO) or nitrated derivatives (-N=O) of benzene or naphthalene, amino hydroxyl groups (e.g., picric acid).
  • Those containing the azo group -N=N- and union-ring derivatives of benzene or anthracene.
  • Derivatives obtained from anthraquinone, from the oxidation of anthracene.
  • Derivatives containing the acridine chromophore =C=N- (imino).
  • Derivatives of imines: oxazine, quinone imine, thiazine.
  • Derivatives of diphenylmethane (auramine) and triphenylmethane (fuchsin) whose chromophore is =C=CH.
  • Xanthene derivatives with variable chromophores.
  • Phthalocyanine derivatives such as Alcian blue or amrillo, which possess a ring resembling hemoglobin.

Auxochrome Groups

• Basic: Groups with cationic groups are responsible for the overall burden of the dye, used to color mainly acidic structures like nucleic acids (charge +) (e.g., hematoxylin).
• Acidic: Those with anionic groups are responsible for the overall burden of the dye. Generally, they stain basic structures contained in cytoplasms (e.g., eosin).
• Neutral: Those that are neither acidic nor basic, but salts (e.g., eosin methylene blue).
• Indifferent: Those that do not possess a defined acid-base character, which is why they usually color tissues by physical impregnation.
• Metachromatic: Most tissues stain the same color as the dye used (called orthochromasia). When using certain basic dyes derived from aniline, some tissue structures are stained a different color; this is called metachromasia, and the stained structure is called a chromotrope.