STAINING OF TISSUE POLYCATIONS BY ANIONIC DYES IN SALT SOLUTIONS WITH SPECIAL REFERENCE TO GINGIVA

Abstract

Over the range pH 5.6-9.4, the presence of KCl or other common metallic chlorides in excess of 0.1 M in 0.05% solutions of Biebrich scarlet or acid fuchsin produced a colored precipitate within 24 hr. Within the same period, over the range pH 2-7, no precipitate resulted when up to 5 M choline chloride (highest concentration tested) was present in 0.01% solutions of these and other anionic dyes used for staining tissue sections. In paraffin sections of frozen-dried tissues, previously exposed to modified Newcomer's fluid or cyanuric chloride in anhydrous reaction medium, equilibrium staining in the presence of increasing concentrations of choline chloride or potassium chloride was investigated at pH 2.28-2.70, 5.6 and 6.18-6.70 using 0.01% or 0.001% solutions of acid fuchsin, Biebrich scarlet or its isomer crocein scarlet, fast green FCF, naphthol yellow S. The tissues studied were: prevertebral structures included in a horizontal section through the cervical region of a mouse, rat abdominal skin and gingiva, human gingiva. In general, increasing salt concentration progressively inhibited staining, but over a limited range could increase staining, e. g., in cartilage, by unmasking additional dye-binding groups through the dissociation of macromolecular polyionic complexes. Sequential staining with the same or two different dyes in presence of different salt concentrations showed that inhibition of straining by salt was not due to elution of reactive material from sections but to competition between salt and dye anions for cationic sites in tissues. The biologic polyanions heparinate and hyaluronate were found to compete with Biebrich scarlet for tissue cationic sites. The concept of critical electrolyte concentration (CEC) proposed for staining with cationic dyes in salt solutions was found to be applicable to staining with anionic dyes and thus to be generally valid for staining that involves polar interactions. Rat gingival keratin, previously shown to have only a low CEC for the cationic dye Alcian Blue, displayed a high CEC for all of the anionic dyes investigated. Some sites in mouse arterial wall and laryngeal cartilage known to have a high CEC for Alcian Blue showed high CEC for Biebrich scarlet. It is suggested that the high CEC of human gingival parakeratin for anionic dyes depends on the presence of arginine, histidine and lysine protein residues and those few α-amino groups capable of protonation.