high background with FITC primary Ab - (Feb/02/2006 )

Hi folks, I am trying to do immunodetection of a cell surface protease in glioma cells. I have a FITC labeled mouse primary antibody and cells that lack the enzyme and transfectants that express it at very high levels. I fix the cells in 4%paraformaldehyde for 20min and permeabilize and block them using 3%heat inactivated fetal calf serum +0,1%triton in PBS for 15min. Afterwards I incubate the slides in the antibody solution (1:50-1:800). I get a high background in the negative cells (more in the nuclei?!) and not a very much higher signal from the transfectants. The dilution of the antibody doesn't make much of a difference.
any suggestions?
Thanks a lot, Busa.

Is it background or auto-fluorescence?
what about cells without antibodies?

Did you use an isotype control? Is it a reliable antibody that you've had positive results with before?

Block with something else besides FCS as perhaps your antibody is having some reaction with the serum and other unspecified contents of the serum. Use 5% BSA instead and dilute your antibody itself in 1% BSA as diluent.