Question about Introcellular antibody staining

Yang WANG doll731 at mail.ku.edu
Sun Nov 13 18:41:53 EST 2005


Hi everyone,

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!

Yang WANG
doll731 at ku.edu
785-864-1842
Department of Molecualr Bioscience
University of Kansas




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