The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets

We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright yellow-green, with maximal excitation at approximately 360 nm and maximal emission at approximately approximately 530 nm. The ELF substrate was used with streptavidin-alkaline phosphatase, to fluorescently label site-specific probes bound to their targets, including cell-surface sites, cytoplasmic organelles, nuclear antigens and cytoskeletal networks. All targets were labelled successfully with both the ELF substrate and fluoresceinated probes or protein conjugates. However, the ELF method was frequently more sensitive, with lower background fluorescence, allowing detection of more lysosomes, actin filaments, microtubules and nuclear targets than were visible with corresponding fluoresceinated probes. The ELF substrate was also used with antifluorescein-alkaline phosphatase to amplify fluorescein signals. We found that the ELF signal was in all cases brighter and more photostable than fluorescein signals, permitting shorter film exposures and allowing more time for examining samples. Surprisingly, relative brightness and photostability depended on the target, rather than being a general phenomenon related to the choice of dye alone.