Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.
A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000037:antagonists & inhibitors; D000818:Animals; D002130:Calcium Phosphates; D002396:Catechols; D002563:Cerium; D002648:Child; D003470:Culture Media; D005260:Female; D006652:Histological Techniques; D006801:Humans; D006861:Hydrogen Peroxide; D007202:Indicators and Reagents; D008297:Male; D010655:Phenylenediamines; D010710:Phosphates; D051381:Rats; D013194:Staining and Labeling
D002130:Calcium Phosphates; D002396:Catechols; D003470:Culture Media; D007202:Indicators and Reagents; D010655:Phenylenediamines; D010710:Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D002563:Cerium; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D006861:Hydrogen Peroxide; D000469:Alkaline Phosphatase; C485829:calcium phosphate, dibasic, anhydrous; C034221:catechol; C029728:4-phenylenediamine; C048083:cerium phosphate