I am examining GR translocation in Jurkat cells upon treatment with dexamethasone(10^-6M). Slides are simple cytospins and not embedded. All papers on the subject suggest largely cytoplasmic staining at baseline, and translocation to the nucleus after 1 hr of treatment. The first time I did the experiment, it worked nicely. However, the next two times I see very nuclear staining at baseline(untreated cells). Has anyone experienced this with GR antibody, or maybe another antibody? Can excessive nuclear staining simply be an artifact of some kind? I use a mouse monoclonal primary and anti-mouse alexa 594 secondary. I have replaced my media to try again, thinking perhaps this was the problem. I don't have results for that yet, though. Here is the general protocol I use for the staining:
Day 1
Day 2
Thanks for any help!
Is there serum in your medium? Serum has endogenous glucocorticoids, use serum free medium or charcoal stripped serum.
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