A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific 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 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.