Background Immunofluorescence - Suggestions on how to reduce non-specific background (Oct/14/2009 )
I'm doing immunofluorescence on human skin samples and am having trouble getting rid of the high background from the secondary AB. The primary ABs are mouse and rabbit, while the secondary AB are goat anti-mouse/anti-rabbit. The samples are blocked in 20% normal goat serum (in PBS) all day. The secondary alone control slides don't show as much background, but it is still present. Any suggestions on how to reduce the non-specific staining?
Have you looked at a completely unstained samples? Some cell types exhibit quite a bit of autofluorescence. Also, check the settings on your camera. If the gain and exposure time are set too high, everything lights up.
The section are paraffin embedded, and I use Histo-Clear instead of Xylene to remove the paraffin when I'm prepping the slides. The second protein, a nuclear protein, I'm staining for turns out much clearer. I have yet to look at section that has not been stained at all for any autofluorescence, but since the secondary alone control is pretty clear I'm confident that is probably not the issue. I've also tried two separate antibodies and am having similar problems. Could it be an issue with antigen retrieval?
Try washing your slides in 0.1 M glycine for several hours- overnight, changing a few times. Usually this sort of background is due to glutaraldehyde left behind in the fixing process.