The Alexa Fluor dyes are a new series of fluorophores, developed by Molecular Probes, that are brighter and more photostable than conventional fluorescent dyes. Recently, I have been using an Alexa Fluor 488 dye-conjugated goat anti-rabbit antibody for immunoflourescencent staining. The Alexa Fluor 488 dye is an extremely bright dye that shows up using the FITC filter on conventional fluorescent microscopes. This secondary antibody has been cross-adsorbed to reduced cross-reaction with other antibodies.
I have been using this secondary antibody at dilutions that range from 1:200 to 1:400 to detect beta-catenin and other proteins in various cell types in culture. Because I was very satisfied by the results I had obtained, I recommended it to my colleagues in the lab and everyone has been extremely happy with the results. The signal is definitely brighter than any FITC-conjugated secondary antibody I have every used (by far) and, as mentioned above, it is much more photostable than FITC even when using the same mounting medium. The signal seems to last even longer when using the Prolong Anti-Fade Kit from Molecular Probes. In addition, the background has been very low and I haven’t had any problems with cross-reactivity with this antibody.
The only issue that I have had with the Alexa Fluor dyes is that their signal can be so bright that it actually leaks out into the DAPI filter. Although I have overcome this problem by using more restricted filters or even using confocal microscopy when possible.
Overall, the Alexa Fluor 488 conjugated secondary antibodies have given me a nice, stable and robust signal. These antibodies are sensitive enough to dilute out fairly far, which is nice because it will save money in the end. I would have no problem recommending these antibodies to anyone.
Mathias Mericskay, PhD
Post-Doctoral Fellow
Mount Sinai Medical Center