Light microscopical localization of enzymes by means of cerium-based methods. V. Optimization of the cerium-lead (Ce-Pb)-technique for alkaline phosphatase.

A modification of the earlier published cerium-based technique for histochemical detection of alkaline phosphatase activity at light microscopical level (Halbhuber and Zimmermann 1985) is described. The reduction of the s-collidine concentration from 200 mmol to 50 mmol, increase of cerium ion concentration rom 1 mmol to 5 or 10 mmol, and sucrose concentration from 7.5% to 15% at increased from pH = 9.0 to 9.5 less than or equal to 9.9 in the incubation medium led to a high intensification of the histochemical reaction. The brush borders of the rat kidney (especially of the epithelial cells of the primary convoluted tubules) and of the enterocytes demonstrate black-brown tinged and precisely localized final reaction products. Moreover, a simplification of the histochemical procedure by employment of postfixed cryostat sections (small intestine) instead of the time consuming perfusion fixed material (kidney) is presented. Several fixatives were also tested. Nakane's periodate-lysine-paraformaldehyde (PLP) or the periodate-lysine-glutaraldehyde (GLP) fixations are superior to the classical glutaraldehyde/paraformaldehyde double fixation. The proposed optimized cerium-based techniques are recommended for a broad use.