A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity

We describe a high-resolution, fluorescence-based method for localizing end ogenous alkaline phosphatase in tissues and cultured cells. This method uti lizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an inten sely fluorescent yellow-green precipitate at the site of enzymatic activity . We compared zebrafish intestine, ovary, and kidney cryosections stained f or endogenous alkaline phosphatase using four histochemical techniques: ELF -97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate couple d with Fast Blue BE (colored) and Fast Red TR (fluorescent) diazonium salts . Each method localized endogenous alkaline phosphatase to the same specifi c sample regions. However, we found that sections labeled using ELF-97 phos phate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activ ity, whereas signals generated using the other methods diffused. We found t hat the ELF-97 precipitate was more photostable than the Fast Red TR azo dy e adduct. Using ELF-97 phosphate in cultured cells, we detected an intracel lular activity that was only weakly labeled with the other methods, but co- localized with an antibody against alkaline phosphatase, suggesting that th e ELF-97 phosphate provided greater sensitivity. Finally, we found that det ecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins.