Fluorescent image of U251 cells stained with AIFM1 antibody. U251 cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated AIFM1 primary antibody (1:200, 2 h at room temperature). For secondary antibody, Alexa Fluor 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Cytoplasmic actin was counterstained with Alexa Fluor 555 (red) conjugated Phalloidin (5.25 mu M, 25 min). Pictures were taken on a Biorevo microscope (BZ-900, Keyence). Nuclei were counterstained with Hoechst 33342 (blue) (10 ug/ml, 5 min). AIFM1 immunoreactivity is localized to the cytoplasm of U251 cells.