Hi all! Question for all you that may be more immuno-experienced than I:
I've consistently run immunofluorescence on pig retina and I seem to have a severe case of autofluorescence/background. I've played around with primary and secondary antibody ratios, but that doesn't seem to help my case. The primary antibodies I use are goat-anti-FLT.1, and mouse-anti-rhodopsin. The secondary antibodies I use are AlexaFluor donkey-anti-goat488 & rabbit-anti-mouse555 (For my experiments, each slide contained only one primary antibody, and it's corresponding secondary. For imaging purposes, I did not double-label on these slides. E.g. FLT.1 was labeled with the 488 secondary, and rhodopsin was labeled with the 555 secondary).I re-hydrate, conduct antigen retrieval, and block as per normal IHC protocol. However, when imaging, I noticed that both slides, labeled with either rhodopsin or FLT.1 seem to "bleed" through to the next filter. For example, mouse-anti-rhodopsin labeled with the 555 secondary works beautifully at a 1:600 ratio. However, when I switch to the FITC filter on my scope, all the retinal tissue appears green on the slides, even though it has ONLY the 555 secondary and NO 488. I've noticed this for the FLT.1 antibody as well (i.e. switch to red filter and tissue fluoresces even though no slide saw the 555 secondary antibody). As I mentioned, I decreased the ratios of all antibodies, but that still doesn't eliminate the problem. If anyone has any ideas as to how I go about fixing this, please let me know. I've only been in the field for a very short period of time, so if I missed something in my description, don't hesitate to ask! Thanks for whatever help you can direct my way! _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet