I need some advice for a problem in Immunofluorescence.
I am interesting in a protein that is expressed mainly in cytoplasm. (as described in many references)
when I carried out Immunofluorescence to stain this protein, I found it was very bright in nucleus in most cells
I have searched this protein in google, and found most of the picture also showed a cytoplasm-located.
my protocols was as follows:
1 Grow cells on coverslips
2 Wash with PBS and fix with 4% PFA 1h at 4 degrees centigrade, followed by wash with PBS for three times
3 block and permeabilize with 0.1% Triton and 3% BSA in PBS for 1h RT
4 Incubate overnight with primary antibody, primary antibody was diluted in 3% BSA-PBS
5 Wash 3 times with PBS; incubate 3 hr RT with secondary; secondary was diluted in PBS
6 Wash 3 times and visualize
Did any procedures in the immunofluorescence affect the location of the protein?
I have seen several posts on here, and I suspect the problem was in the fixation or the permeabilization. was the time too long?
could anybody shed light on this?
Any advice would be greatly appreciated!