The Alexa Fluor 568 goat anti-mouse antibody is the secondary antibody dedicated to those who have always found that rhodamine signals are too weak and diffused to yield good immunofluorescent photos. The Alexa Fluor 568 dye is a rhodamime substitute from a new category of fluorophores developed by Molecular Probes. These fluorophores are brighter and more photostable than conventional dyes. Molecular Probes has taken these new fluorophores and conjugated them with secondary antibodies, in this case goat anti-mouse antibodies, that have been cross-adsorbed to reduce the amount of cross-reaction that could occur with the primary antibody.
Our lab has used the Alexa Fluor 568 goat anti-mouse IgG at dilutions from 1:200 to 1:300 in PBS/5% goat serum in immunofluorescence assays to detect desmin, SM alpha-actin and other proteins in various cell types in culture. Everyone in the lab has been very satisfied with the results. The red fluorescent signal from Alexa Fluor 568 is visible using the rhodamine filter on a conventional microscope and it is definitely brighter than any rhodamine-conjugated secondary antibody I have used so far. The background that I have seen using this secondary antibody is low, it does not appear to cross react with any other primary antibodies (i.e. rabbit polyclonal antibodies used in for double staining specimens) and it is definitely much more photostable than rhodamine. The signal lasts much longer when using the Prolong Antifade Kit, also from Molecular Probes.
The only problem that I have with the Alexa Fluor 568 conjugated secondary antibodies is that the signal can sometimes be so bright that it actually leaks into the DAPI filter. This relatively amazing considering the distance between the excitation length of the rhodamine filter and the DAPI filter. But this problem can be dealt with by increasing the dilution of the secondary antibody and it does not create any problems when using a confocal microscope.
All in all, I would have to say that the Alexa Fluor 568 conjugated goat anti-mouse antibody is a a very good secondary antibody that gives a nice, stable and
Mathias Mericskay, PhD
Mount Sinai Medical Center
Molecular Probes' Alexa Fluor® 568 Secondary Antibodies
Gives a bright, stable signal with little-to-no cross reactivity
Watch out for the signal leaking into the DAPI filter
The Bottom Line
It’s a very good secondary antibody that gives a clean, robust and stable signal