The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH

Abstract

SUMMARYWe used the ELF-97 ( E nzyme-L abeled F luorescence) phosphatase substrate, 2-(5 Ј -chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at ‫ف‬ 360 nm, with emission centered at ‫ف‬ 530 nm. This large Stokes shift allows ELF-97 signals to be easily distinguished from sample autofluorescence and signals arising from counterstains or other fluorophores. The ELF-97 precipitate was extremely photostable compared to fluorescein, allowing multiple photographic exposures of samples without significant signal intensity loss. For RNA in situ hybridization, labeling was specific and localized well to targets in cultured cells, tissue sections, and whole-mount zebrafish embryos. ELF-97 signals developed in seconds to minutes and were easily distinguished from pigmented tissues or cells, unlike those obtained using colorimetric substrates. We used the substrate with singly biotinylated short oligonucleotides to detect actin mRNA in MDCK cells and actin and ␤ -galactosidase mRNA in LacZ ϩ mouse fibroblasts. We also used a biotinylated cDNA, complementary to the mRNA encoded by the constant region of the T-cell receptor ␤ -chain, to specifically identify T-cells in mouse lymph node tissue sections. With digoxigenin-labeled probes, we detected several developmentally expressed mRNAs in whole-mount zebrafish embryos. Hybridization to centromere repeat regions in human metaphase and interphase chromosomes was also detected; ELF-97 signals were manyfold brighter than signals obtained with fluorescein conjugates. Finally, Southern blot hybridization using singly labeled oligonucleotide probes yielded a sensitivity similar to that obtained with radioactivity.( J Histochem Cytochem 45:345-357, 1997 )

Enzyme Cytochemical Techniques for Metabolic Mapping in Living Cells, with Special Reference to Proteolysis

“…A unique fluorogenic substrate for phosphatases, 2-(5 Ј -chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone or ELF-97-phosphate, has recently been developed (Paragas et al 1997). The ELF-97-alcohol that results after liberation of the phosphate group by phosphatase activity forms a bright yellowgreen fluorescent precipitate at the site of the activity (Figure 2).…”

A High-resolution, Fluorescence-based Method for Localization of Endogenous Alkaline Phosphatase Activity

“…The ELF-97 alcohol liberated from the substrate forms a bright yellow-green fluorescent precipitate at the site of enzymatic activity, which is directly visible by fluorescence microscopy (Singer et al 1994). Previous work has employed the ELF-97 phosphate in enzyme-mediated labeling techniques including immunohistochemical (Larison et al 1995), cytochemical (Brumbuck and Wade 1996;Paragas et al unpublished observations), and mRNA in situ hybridization (Bueno et al 1996;Currie and Ingham 1996;Jowett and Yan 1996;Paragas et al 1997) applications. The ELF-97 precipitate was found to be more photostable than fluorescein and distinguishable from other fluorescent labels, sample autofluorescence, and pigmentation.…”

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…On the other hand, in ELF, target RNA is also first hybridized by primary-antibody-conjugated nucleic acid probe, and then secondary-antibody-conjugated phosphatase is further attached. After that, ELF-97 (enzyme labelled fluorescence) phosphatase substrate 90 is added, which is converted to brightly and stably fluorescent ELF alcohol in the presence of phosphatase. Using ELF amplification, individual miR-15a and miR-155 were identified as bright and photostable fluorescent spots in single cells 11 .…”