u/tgegz

Any ideas on his-tag immunofluorescence staining in HEK293T cells? Both my non-transfected control and experimental cells are positive for His tag. I have tried range of primary antibody dilution (1:500 - 2000) and the signals are still present in my negative control. My staining protocols includes fixing in 2% PFA for 10 minutes, permeabilization in 0.2% triton x-100, blocking in 2% BSA, incubation in primary and secondary antibody for an hour each (with the appropriate wash steps).