Light microscopical localization of enzymes by means of cerium-based methods. I.V. Optimization procedures for acid...

The earlier described cerium based histochemical reaction for acid phosphatase [Ce-Pb-reaction, Zimmermann and Halbhuber (1985)] was optimized. The target tissues (kidney, intestine) were fixed by perfusion with glutaraldehyde in cacodylate or piperazine buffer in anesthetized animals. Postfixation of prefixed sections is not advantageous because of the detectable repressing of the enzyme activity. Moreover, the employment of unfixed cryostat sections, which were postfixed, was always connected with a complete abolition of the acid phosphatase activity. The optimal concentration of the primary capture cerium III chloride in the incubation medium is about 1 mmol. Lower concentrations lead to an incomplete histochemical detection of phosphatase activity in lysosomes. The treatment of cryostat sections of perfusion fixed tissue with borohydride (diminution of aldehyde induced cross links) or with dimethylsulfoxide (extraction of lysosomal materials or the well known vehicle property) brought about an improvement of the penetration capacity for cerium-III-cations into the target structures. After conversion of the cerium phosphate (primary specific reaction product) into cerium perhydroxide, oxalate or fluoride, the Ce-Pb-reaction was negative. Therefore, these blocking reactions represent specific inhibition controls, which indicates the formation and presence of cerium phosphate. On the basis of these reactions it is possible to check the specificity of the histochemical Ce-Pb-reaction for phosphatase activity in sections.