The chemical and histochemical properties of Alcian Blue

Summary

Blocking and unblocking reactions have been applied on cartilage and epithelial mucins in order to ascertain their specificity and their effects in Alcian Blue staining.

Methylation, followed by saponification, acetylation and deacetylation and deamination were all investigated. Blocking of tissue polyanions by CPC and unblocking by KCl or MgCl₂ have been studied. Finally, pepsin treatment was applied along with the bromphenol blue stain for the detection of positively charged protein.

The findings indicated that in tracheal cartilage the highest AB uptake occurs only after deamination, thus indicating that the accessibility of the dye to the reactive tissue radicals is prevented by a protein component. In rat joints the AB staining was always intense and little or no improvement in the staining reaction was obtained after the destruction of the amino groups. These data suggested that in rat joints protein interference in the dye-polyanion binding was negligible. The bromphenol blue reaction demonstrated, in fact, that the distribution of the positively charged protein groupings (presumably amino groups) was much higher in tracheal than in epiphyseal cartilage.

Acetylation did not affect tissue alcianophilia with the exception of human fetal trachea, in which deacetylation restored AB staining.

Methylation gave rise to different results according to the temperature used, to the length of time of the reaction and to the pH of the dye solution. Differences were also noticed depending on the tissue involved in the reaction. From the findings reported in this work some reservations should be made on the selectivity and the specificity of methylation as a means of recognizing chemical groupings.

The blockade of acidic radicals in the connective tissues by means of quaternary ammonium salts is considered a selective and reproducible method. The use of KCl and/or MgCl₂ displaced CPC and restored alcianophilia and metachromasia as well.