Immunofluorescence Detection of p53 Status

The cells were seeded on a coverslip (Corning Life Sciences, United States) and then incubated at 37°C overnight. Subsequently the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The cell membrane was permealized with 0.5% Triton X-100 for 5 min to allow the entry of antibody into cells. Then the permealized cells were blocked with 1% BSA (bovine serum albumin) for 1 h at room temperature, followed by incubation with the primary antibody PAB1620 or PAB240 (Abcam, United States) at 4°C overnight. These antibodies can recognize the p53 protein status. PAB1620 and PAB240 are able to specifically recognize wild-type and mutated p53, respectively (Yu et al., 2012 (link)). Thereafter, the cells were incubated with the goat anti-mouse IgG Alexa Fluor 488 secondary antibody (Invitrogen, United States) for 1 h at room temperature. The nuclei were stained with DAPI (4′, 6-diamidino-2- phenylindole) for 10 min at room temperature. After washes with PBS, the immunofluorescence of cells was detected by laser scanning confocal microscope LSM700 (Zeiss, Germany).