A NEW ONE-STEP METHOD FOR THE HISTOCHEMISTRY AND CYTOCHEMISTRY OF CA^<2+>-ATPASE ACTIVITY

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A new one-step lead citrate method is introduced for the histochemical and cytochemical demonstration of Ca^<2+>-ATPase activity. The standard incubation medium contains 3 mM ATP as the substrate, 10 mM CaCl_2 as the activator, 2 mM lead citrate as the capture reagent, and 250 mM glycine-NaOH or-KOH buffer, pH 9.0. The incubation medium was clear and stable. Cytochemical reactions in this incubation medium were examined in various tissues; liver, kidney, heart, small intestine, trachea, bone marrow, and femoral muscle. The reaction products were found in plasma membrane, mitochondria, myofilaments, axoneme of cilia, sarcoplasmic reticulum, tramsverse tubules, gap junctions, lysosomes, and others. The cytochemical reaction showed Ca^<2+> and substrate dependency. Preheating sections in boiling water resulted in complete loss of the reaction, but pretreatment with 10 mM EDTA or EGTA did not affect it. Inhibitors such as 10 mM PCMB, 0.1 mM quercetin, and 0.1 mM oligomycin, partly reduced the cytochemical reaction in some tissues, but 0.5 mM bromotetramisole left it unchanged. Substrate specificity was proved by the substitution of various substrates. Reaction products were found to be composed of lead phosphate with X-ray microanalysis. From these results, it was concluded that the cytochemical demonstration of Ca^<2+>-ATPase activity was successful by this method at both light and electron microscopic levels.
日本組織細胞化学会の論文