Use of a new fluorogenic phosphatase substrate in immunohistochemical applications

We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.