Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.