We describe a fluorescent histochemical technique for detection ofnonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 mm in the stain solution. The stain solution itself proved to be non-fluorescent, thus

The cell surface alkaline phosphatase (APase) of the preimplantation mouse embryo has been characterized in situ by inhibition studies and fluorescent histochemistry. The embryonic APase has also been compared in the inhibition studies to the APase expressed on mouse F9 teratocarcinoma cells and, in some instances, to the APase of mouse intestinal epithelial cells. The embryonic APase was active over the pH range of 6.0-10.0, with the optimal pH for full activity in the range of 8.5-10.0. The embryonic APase was remarkably heat stable with significant loss of activity detected only after a 1 h incubation at 90 degrees C. A variety of specific and nonspecific APase inhibitors were applied to embryos to determine the nature of the APase isozymes expressed on these cells. The embryonic APase was totally resistant to levamisole, tetramisole, bromotetramisole, and L-homoarginine. The embryonic APase was inhibited by L-phenylalanine in a concentration-dependent fashion and by sodium arsenate, sodium vanadate, ethylenediamine tetraacetic acid, and slightly by 1,10-phenanthroline. The inhibition profile of the mouse embryonic APase, therefore, resembles most closely that reported for human placental APase with respect to heat stability and that reported for mouse intestinal APase with respect to inhibitor sensitivity.