I guess this is a basic immunostaining question. I am preparing cells for flow cytometry. First I'm staining for a pluripotency marker, then I'm staining for BrdU.
I add 100 uL of SM (2%FBS, NaN3 in 1xHBSS), then 80% EtOH and incubate at 4*C for 30 minutes or more. Then I permeabilize with BD cytowash/perm buffer (a saponin based buffer used to permeabilize cells) and then I incubate O/N with an antibody against the pluripotency marker. When I retreive the cells in the morning, the cells become one giant mass that cannot be dissociated into single cells. I can no longer proceed with the rest of my staining.
Does anyone know what could be happening? I would love to know peoples thoughts on this
Thank-you in advance!
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Immunostaining for flow cytometry
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