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

Abstract

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-H2O2 reagent. 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 *1 Details have been presented at the 83rd Annual Session of the Japanese Assocition of Anatomists,