Dear All

I want to perform immunofluorescence in S2 cells. However, this I want to do after RNase A treatment of the cells, therefore I need to permeabilise the cells before fixation. I tried some protocols which use either Triton X-100 (0.05%) or NP40 (0.05%) or a combination of both. However, these protocols are causing damage to the cellular architecture. In fact, I tried very small concentrations of these detergents and performed several time points from 1 sec to several minutes using 0.005% Triton and yet the cells look increasingly damaged as the incubation increases. I read that Digitonin, another nonionic detergent should be a bit milder that the above two, however, could no find any protocol where it was used on S2 cells.

Any suggestions and help would be greatly appreciated.

Thank you

Ikram