Hi Natalia, What type of tissue are you working with, and how was it processed? Are these paraffin sections or cryosections? What fixative did you use? These details can help narrow down a potential problem. If you post your protocol that will help.
You might be seeing autofluorescence from the tissue itself, especially if you are working with paraffin sections or formaldehyde-fixed samples. See what the tissue looks like with no secondary antibodies - if you see the same fluorescence, you have autofluorescence coming from the tissue itself. You will need to either process the tissue differently, try to block the autofluorescence, or amplify the signal of your antibody staining above the level of the autofluorescence (using avidin/biotin or TSA for example). To check for nonspecific binding of the primary or secondary, you can run a panel of stainings, leaving out the primary antibody or replacing with a general isotype control, as Kim suggested. Andrea Marion Graduate Student University of Illinois at Chicago Hi everybody! Maybe you can help me. I'm trying to do immunofluorescences to see the colocalisation between two proteins (PSD + Oligomeres) using Oregon Green (goat anti mouse) and Texas Red (goat anti rabbit). But my probleme, is that the analysis of the slides reveals too much colocalisation. I mean, all seems yellow, as if both fluorochromes show the same thing. It's the first time I do fluorescence, so I don't know where could come the problem. I tried to do a simple staining, 1 slide with my first antibody and its fluorochrome, and the second slide with the other first antibody with its fluorochome and then check the result with the filter red and green. But it's strange because I see both color as if I had put both fluorochromes (second antibodies). Has somebody already had this problem? What can I do to resolve it? Thank you very much for your help. Natalia _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet