Ultracytochemistry in Sections Osmicated and Embedded in Epoxy Resin and Chemical Analysis of Chelated Zinc on Cytochrome c Adjective Reaction

A histo- and cytochemical method called the cytochrome c adjective reaction was devised to detect sulfated mucopolysaccharides following experimental results, which showed that cytochrome c intensely oxidizes leucopatent blue (LPB)hydrogen peroxide (H2O2) solution and changes it to its original blue color (in light microscopy) or electron dense deposits or patches (in electron microscopy). Ultrathin sections were first treated with 0.1% acetone (50%) solution of cytochrome c, washed in Walpole's buffer at pH 3.0 and then reacted with LPB-H2O2reagent. This reaction selectively stained or detected cartilage matrix without any electron staining, especially in lacunar borders (territorial matrix). This demonstrates that the reaction will not succeed unless sections are treated with cytochrome c before the histo- or cytochemical reaction is employed. Incidentally, the molecular ratio of residual zinc against residual dye was estimated at ca.1 by both atomic absorption spectrophotometry and the chelate titration method. Judging from these analytical results, the chemical formula of LPB was postulated as a chelate complex formation of leuco-dye with zinc, which can be clearly demonstrated using the adjective cytochrome c reaction with an electron microscopy. These ultracytochemical and analytical findings suggest that the dye should be reduced by catalystic acid zinc, and the same time, added zinc should be chelated into the leuco-dye molecule. Thus, this technique furnishes an ultracytochemical method for the localization of chondroitin polysulfate in the lacunar border of cartilage matrix.