Primarily the cytochrome c-leucopatent blue-hydrogen peroxide (CC-LPBH
2O
2)reaction intensely stained the matrix of cartilage, substantia propria of the cornea, and mast cell granules, and occasionally stained the nuclei of certain epithelial cells. In the nuclei, non-specific combination of CC with various components was dissociated by treatment with buffer solution of pH 3.0. After methylation, the reactions of mast cell granules(37°C), substantia propria of the cornea(37°C)and cartilage(60°C)were entirely abolished. In sulfation sections, the reactions of mast cell granules were completely reduced, while those of goblet cells, collagen fibers and arterial wall were markedly enhanced. By digestion with novel hyaluronidase, the reactions of umbilical cord and cockscomb were abolisned, while mucopolysaccharides containing sulfuric acid esters were completely uninfluenced except in the case of the cornea and sclera. In digestion tests with chondroitinase ABC and AC, mast cell granules were completely uninfluenced, while cartilage and mucous acinous cells of the lingual glands retained their staining affinities with CC-basic proteins to some extent. In the critical electrolyte concentration method for distinguishing individual acid mucopolysaccharides, the staining pattern shifted and became strikingly reduced compared to the Alcian blue method. Mast cell granules and tracheal cartilage, however, were clearly stained at 0.2 M MgCl
2in CC-adjective solution in contrast to staining of the cornea at 0.05 M MgCl
2. The CC-adjective reaction is thought to provide a method for the localization of polyanions such as sulfate, carboxyl and phosphate groups. In the specific reaction at pH 3.0, however, its staining property generally runs parallel with the acidity and density of the polysulfated mucopolysaccharide. Further, data obtained by histochemistry were in agreement with those obtained by electrofocusing analysis of CC-heparin complex in vitro.
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