Thread: Indirect immunofluorescence fixation and permeabilization

I am working with a human endothelial cell line where I am observing for localization of what is hypothesized to be a transmembrane protein. The primary antibody I am using binds to the C-terminal epitope, that is expected to be localized in the cytosolic region. In previous experiments, I have captured indirect immunofluorescent images which appear to be nonspecific interactions of primary antibody to permeabilized cells. I believe that I am efficiently washing my cells after incubation with the primary antibody. My guess is that I am not effectively permeabilizing the cells; I have used 3.7% paraformaldehyde solution followed by a blocking solution containing 0.1% Tween-20.

I have read an ab-cam protocol that suggests to also use methanol or acetone to fix and permeabilize the cells. Does anyone have experience or other suggestions with permeabilizing human cell lines for indirect immunofluorescence imaging?