Jennifer,
You Wrote: I work with a scientist who insists on using primary antibodies directly conjugated with FITC or Texas Red for IHC. In my past experience these directly conjugated antibodies didn't give a strong enough signal for use in IHC (I've seen them used only for FACS analysis). I would appreciate your professional comments. Jennifer M. Anderson, Scientist Halozyme Therapeutics, Inc. 11404 Sorrento Valley Road San Diego, CA 92121 858-704-8333 If your antibody is conjugated to a fluorophore, as you described, you may run into the problem where the FITC and Texas Red molecules are in too close proximation to each other and will "quench" due to a trading of electrons between the FITC molecules when these are excited. This is a problem we had with murine CD4-FITC and murine CD8-FITC. The result is the antibody binds to the antigen, a protein, but the quenching causes the FITC to not fluoresce. Quenching is a NOT photobleaching aka "fading", but a different physical chemical happening that "reduces the excited state lifetime, and the quantum yield of the affected fluorophore." You can go to www.olumpusconfocal.com/theory/fluorphoresintro.html and read about this under Quenching and Photobleaching (two different things). After long discussions with my Physical Chemist husband who works with quenching studies on fluorescent molecules, we do not use direct fluorophore conjugated antibodies in the lab for staining tissue sections anymore. The Jablonski Diagram also shows this. It will not happen with all antibodies that are conjugated to fluorophores due to their spatial relationships (in terms of nanometers) but if it does, Ray Koellings suggestion to come back with an antiFITC or Anti Texas red should solve the problem. We are better served by having the primary antibodies biotinylated, then come back with a Streptavidin Alexa fluor dye (488 for FITC, and 594 for Texas Red). _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet