Axiovert 25 fluorescent microscope

HeLa cells were seeded onto sterile coverslips in a 6-well plate at 30–40% confluence. The next day, plasmid transfection was performed as indicated. At 24 h post-transfection, the cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. After blocking for 30 min with blocking buffer (1% BSA in PBS buffer, pH 7.4), the cells were incubated with Abs against Myc or p65 for 1 h and then with a fluorophore-conjugated secondary Ab for an additional 1 h. The cell nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) for 5 min. The coverslips were sealed onto the glass slides using nail polish, and the slides were then subjected to fluorescent microscopy analysis. The images were taken with a Zeiss Axiovert 25 fluorescent microscope.