BrdU immunofluorescence protocol - (Nov/02/2012 )
I am trying to study cell proliferation using BrdU immunofluorescence. I use control cells which I know are proliferating obtaining no results. I think it is because of BrdU concentration (I have read that I can only use 10microM). Have anybody try with higher concentrations?
I have used 10uM and it worked well. How long are you incubating the cells with the BrdU? You could try incubating with the BrdU for longer to increase the signal.
I incubate the cells for 4 hours, but as I saw nothing I increase to an overnight with the same results.
Are the cells confluent? If they're confluent or close to it, they won't be proliferating and the BrdU assay probably won't work. Have you tried lowering the number of cells plated? Also maybe try increasing the amount of anti-BrdU antibody used and double checking that your reagents are still good.
The cells were not confluent and I use a really low dilution of the antibody. Anyway it still don't work!
I will try to incubate with BrdU overnight. Anybody have any extra advice?