Fixation for IF??? - best method for nuclear antigens (Sep/06/2005 )
I am having trouble with attaining consistent staining using ice-cold methanol fixation. So, I have a couple basic questions:
1. is methanol sufficient for visualization of nulcear antigens?
2. if I use paraformaldehyde do I have to permabilize the cells afterward?
Methanol and acetone based fixatives are not suitable for immunofluorescence of nuclear antigens. Incubate in 2% PFA in PBS for 15 min at RT followed by a PBS wash. Are you working with frozen sections or cell cultures? If cell cultures, yes you will need to permeabilize afterwards. What type of detergent is somewhat up to you but I use 0.1% Triton X 100 for 5 minutes on a rocker at room temperature with good results.