Protocol Online logo
Top : New Forum Archives (2009-): : Flow Cytometry

Intracellular staining, isotype control not stable - (Jan/22/2013 )

I have some problems with intracellular staining. Im fixating my cells with pfa, and permeabilizing them with saponin, and use an isotype control mouse IgG1 FITC, the same as the specific antibody. However the isotype control is so reactive and as the cells are stimulated the percentage of isotype positive cells increase by 30 %. But the specific antibody just increase with 5 %. and shows much less positive cells than the isotype control. This is so frustrating!
I also tried another pair of antibodies, rat IgG1 PE, but it shows the same tendency. Any advice?


Maybe it's a kind of unspecific binding... Which cell type is it, by the way? Depending on that you might try blocking Fc-receptors. Have you tried different dilutions of the isotype control ? Maybe a higher dilution will give a better result. How many times do you wash ? You might try to increase the number of washings too.