USE OF A NEW FLUOROGENIC PHOSPHATASE SUBSTRATE IN IMMUNOHISTOCHEMICALAPPLICATIONS

We used the phosphatase substrate o-2'-phosphoryloxyphenyl)-6-chloro-4 -[H-3]-quinazo with standard alkaline phosphatase-mediated immunohisto chemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzym e-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at similar to 500-580 nm (bright yellow-green). We sho w that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labe led secondary reagents. The staining produced with the ELF substrate i s much more photostable than that produced by fluorescein-labeled seco ndary reagents, thus allowing time to examine, focus, and photograph t he ELF-labeled tissue under high magnification. Moreover, the ELF prec ipitate exhibits a Stokes shift of greater than 100 nm, a characterist ic that has enabled us to overcome the problem of distinguishing signa l from background in this autofluorescent tissue, in addition, we show that the ELF product's large Stokes shift makes the ELF substrate ide al for multicolor applications.