Question about Introcellular antibody staining
doll731 at mail.ku.edu
Sun Nov 13 18:41:53 EST 2005
I am staining embryonic stem cells using different introcellular antibodies as
differentiation marker, however, they all showed positive for over 95% , which
can't be true. When I set FSC threshold to 0, a negative population showed up,
whose FSC-SSC both are near to zero.I used 7-AAD as a viability dye, that
negative population is negative for 7-AAD. Other than that, all the population
showed a much higher fluorescence intensity compared to negative control.
Here is my direct staining protocal.Could anyone help me figure out what the
problem might be?
1.Fix w/ 4%PFA 30min on ice,wash;
2.Permiablize w/ 100% methanol drop by drop, incubate on ice 30min;
3.rehydrate with PBS 5min twice;
4.block w/ 1%BSA+3%Rat/mouse serum(depending on antibody species) 30min RT;
5.Incubate antibody 1hr RT.
6.Wash 10min twice.
Thank you for your attention!
doll731 at ku.edu
Department of Molecualr Bioscience
University of Kansas
More information about the Cytometry