Immunofluorescence

C3H10T1/2 cells were seeded onto sterile coverslips. After 24 hr, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed twice with PBS, and then blocked with 5% BSA in PBS for 20 min. Coverslips were rinsed with PBS and incubated overnight with mouse Alexa Fluor 488 anti-HA antibody (BioLegend, 901509) at a dilution of 1:100. Coverslips were then washed five times with PBS and samples stained with DAPI (4′,6-diamidino-2-phenylindole). Fluorescent images were obtained with a Zeiss fluorescence microscope.