Enzyme-Labeled Fluorescence 97 (ELF-97) is a new substrate developed by Molecular Probes for alkaline phosphatase (AP) which is widely used as a reporter gene in various applications. ELF-97 is a soluble, weakly blue-fluorescent, phosphorylated molecule that yields a bright yellow-green fluorescent precipitate upon cleavage by the AP enzyme.
The ELF-97 kit was designed for detecting endogenous AP activity in tissue sections but because the kit consists of the substrate, a dilution solution and a mounting medium, you can use it for any application where AP is involved. I have used it to detect human placental AP expression in retrovirally-infected primary cells and transfected stable cell lines (the retroviral vector I used carries a human placental AP cDNA as a reporter gene). Using the ELF-97 kit, I have consistently obtained specific, bright fluorescent precipitates in less than 90 sec after heat inactivation of the endogenous AP activity. The signal is generally very intense and, in my experiments, consisted of well-defined bright green spots along the cytoplasmic membrane and in the cytoplasm itself. To observe the signal, I used a DAPI filter on a conventional fluorescent microscope - but Molecular Probes also offers a set of filters that are designed specifically for this stain. However I don't think these special filters are required for most applications because, in my hands, the signal did not leak out in the FITC or Rhodamine filters, despite its brightness.
In addition to being extremely bright, the ELF-97 stain was clearly more specific and gave higher resolution than conventional AP stains. When I used an NBT/BCIP purple-blue colorimetric substrate on duplicate plates, I noticed that the NBT/BCIP precipitate tends to diffuse to adjacent cells and localizing the signal in a specific part of the cell was difficult. As mentioned above, the ELF-97 signal can be visualized with a conventional fluorescent microscope using a DAPI/Hoechst filter. Moreover, I was even able to see the signal under bright field illumination, which allowed me to see both the cell morphology and the ELF-97 precipitate. I also found that the signal was extremely photostable when I used the mounting media that comes with the kit. And, in contrast to the conventional NBT/BCIP substrates, the reaction time for the ELF-97 was fast – you get your results in a few minutes instead of an overnight incubation.
This last point, however, can sometimes be a problem - the reaction can almost be too fast. Molecular Probes suggest that you perform the reaction under the microscope, but this is not always convenient for many reasons (the area around the microscope is too crowded, far from your own bench, etc.). Also, one has to be aware that the time to localize and clearly recognize positive cells may vary from one sample to the other, introducing variations in reaction time. Personally I have chosen to perform the reaction for 90 seconds on my bench for all samples - but some tests that I have performed letting the reaction go for 3 minutes still gave me good specific staining with no background.
In summary, I think the ELF-97 kit is a good product that is more specific and more sensitive than conventional AP substrates. It is easy to use and does not require a lot of testing to optimize the reaction conditions. The results are robust and this kit can be combined with other fluorophores to perform multiple stains on a given sample.
Mathias Mericskay, PhD.
Post-Doctoral Fellow
David Sassoon's lab
Mount Sinai Medical Center, NY