I've been trying to perform immunofluorescence on HEK293 cells overexpressing HA-tagged proteins.
I tried anti HA 12CA5 (Roche) 1:100, anti HA (C29F4) rabbit mAb from Cell Signalling (1:1000 and 1:500) and anti-HA (6E2) mouse mAb from Cell signalling (1:100).
I performed fixation with PFA 4% for 15 min at rt, then I blocked with 5% NGS, Triton 0.3% in PBS for 1 h at RT. The incubation with the primary antibodies was done either overnight at 4 degrees or overnight at rt, followed by incubation with secondary antibodies (Alexa Fluor conjugated from Invitrogen) for 2 h at rt. I used Triton 0.3% also during the washing steps.
The antibody from Roche doesn't seem to work at all, while the others give a very faint signal.
I also checked the expression of my constructs by Western blot and they are definitely expressed.
Does anyone have experience with these antibodies or any tips to improve the staining?