Rate factors in staining by Alcian Blue.

Abstract

Alcian Blue stains much more slowly than other basic dyes, and differential staining by the dye is greatly influenced by differences in the rate of staining of various basophilic components. Epithelial mucin stains most rapidly, followed by mast cell granules, pericapsular cartilage matrix, interstitial cartilage matrix, nuclei and cytoplasmic chromidial substance (presumably RNA), in that order. Sites containing nucleic acid stain intensely under some conditions, e.g. after some days in Alcian Blue at pH 4 and room temperature and, therefore do not lack affinity for the dye. Nucleic acids are not, however, in general easily stained with Alcian Blue, being extremely slow-staining and also being easily leached from the section by hot or acidic solutions. Not only do most substrates fail to reach a plateau value of dye uptake after ‘normal’, relatively short, staining times (e.g. some hour), but the reversibility of staining by Alcian Blue is markedly affected by the duration of staining. The dye is largely or completely irremovable from most substrates after any except the shortest staining times, but cartilage matrix and mast cell granules are exceptional in that a good deal of dye is removable from these substrates by 1.0m MgCl2, even after prolonged staining. Raising the temperature of, or adding urea to, the dyebath decreases dye aggregation and increases the uptake of Alcian Blue, particularly by relatively slow-staining substrates. Mast cell granules stained with hot dye under some conditions (e.g. 0.05% dye, pH 4, 56°C, 18 hr), but not with cold dye (e.g. 7 days at 4°C) unless pre-treated with hot buffer. The aggregation of Alcian Blue in solution, as assessed by changes in the absorption spectrum, is markedly affected by dye concentration, dyebath temperature, additives such as salt or urea, and also by the age of the solution, the dye being notably unstable under some conditions. The ‘critical electrolyte concentration’ method of Alcian Blue staining, for the characterization of tissue polyanions, theoretically requires staining to be carried to a true (reversible) equilibrium. The present findings throw doubt on the validity of the method.