I am testing translocation of protein from the cytoplasm to the nucleus, I've got a lot of background nuclear fluorescence despite that I used 0.3M glycine in the blocking buffer.
Wash fixed cells with pbs and before cytospinning the cells wash also with serumfree pbs.
You can also block slides with PBS with 10% FCS for an hour. that works for my AML cells.
Thank you John for your comment, I am washing my cells with PBS after 4% PFA fixation three times and with 0.3% tween in PBS after the primary and the secondary AB incubations also three times each. However, I am using tube method and centrifugation in all these steps. Do you think this can be a cause for that? What do you mean by serum free PBS, the PBS I am using contain no serum. Thanks again.
Just normal PBS, you could also try without tween.
That could also help.
Decrease your secondary concentration by half and increase your wash times 30%. Also consider using a high quality anti-fade - it seems kind of backwards to add something to increase your fluorescence but my experience is that it seems to just increase the integrity of target signals and lower background. Be sure your using fresh PFA and your sells are not contaminated too. If there's myco on the slide is bleaches out everything and actually causes background in other (non-myco) areas to increase.
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