Halbhuber K J, Hulstaert C E, Gerrits P, Möller U, Kalicharan D, Feuerstein H
Department of Histochemistry, Friedrich Schiller University Jena, Germany.
Cell Mol Biol. 1991;37(3):295-307.
A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.
描述了一种用于大鼠肾组织半薄和超薄塑料切片（Epon 812、Lowicryl K4M、乙二醇甲基丙烯酸酯）中磷酸铈（Ce III）的新可视化（Ce/Ce-H2O2-DAB-Ni）方法，这些切片在包埋前已进行孵育以显示磷酸酶（碱性和酸性磷酸酶、5(1)-核苷酸酶、Mg依赖性ATP酶）。为此，在NaOH-乙醇溶液中去除疏水性Epon树脂，而亲水性Lowicryl和甲基丙烯酸酯切片不需要任何蚀刻。初级反应产物Ce III-磷酸盐在Ce III-柠檬酸盐溶液中放大，随后用H2O2氧化，然后在含有DAB-镍的介质（Ce IV-过羟基诱导DAB聚合原理）中可视化。该方法产生了非常清晰的酶活性定位。在0.5 - 2.0微米半薄切片中的最终反应产物（DAB-镍聚合物）为蓝黑色；完全防止了背景染色。通过用OsO4后处理（形成锇黑）获得了染色对比度的增加。此外，通过银增强可以在40纳米厚的超薄切片中显示酶反应产物，这利用了聚合的DAB-镍络合物的高嗜银性。该程序取代了早期发表的技术。
