抄録
A new histochemical staining meth o d called cytochrome c adjective reaction was devised for the demonstration of acid mucopolysaccharide from the experimental results that cytochrome c purified from horse heart intensely oxidizes leucopatent blue-H2O2 solution and changes it to original deep blue. Tissue sections in this reaction were first treated with 0.1% aqueous solution of cytochrome c, washed well in distilled water or formate buffer at pH 3.0 (in electron microscopy), then stained with a peroxidase reagent such as leucopatent blue or benzidine solution containing hydrogen peroxide. This reaction selectively stained the matrix of cartilage, substantia propria of cornea and granules of tissue mast cells, while tissues containing carboxylated mucopolysaccharides remained unstained. Thus, this technique furnishes a method for localization of sulphated mucopolysaccharides such as chondroitin sulphate, keratan sulphate and heparin, as shown in experimental results obtained by the comp lex formation in vitro and in tissue sections of cytochrome c with acid mucopolysaccharide, by paper electrophoresis of the complex, and by enzymatic digestion tests in tissue sections. Further, the mechanism of this c y tochrome c adjective reaction is postulated to be a complex formation of an amino group of cytochrome c with a sulphate group of acid mucopolysaccharides, whic h can be clearly demonstrated by peroxidase reagents at the level of light and electron microscopy. Never t h eless, it is desirable to combine more sensitive reagents with cytochrome c to detect all acid mucopolysaccharides. Furthermore, the factors that disturb the staining of carboxylated mucopolysaccharides in this reaction still remain unanswered. We would like to thank Dr. Kiy o shi Sekita, Professor of Biochemistry, School of Medicine, Keio University for helpful suggestions and encouragement. We are very grateful to Dr. Hiroshi Kushida and Mr. Kunio Fujita in whose laboratory part of this work was planned and performed.
