p53 immunofluorescence

Cells, fixed as described above, were washed with cold PBS 1X, permeabilized with cold PBS 1× with 0.5% Triton X‐100 for 20 min at 4°C, incubated in blocking solution (1% BSA and 5% FBS in PBS 1X) for 30 min at RT and then treated with p53 specific antibody (1:50, Santa‐ Cruz) in PBS 1× overnight at 4°C. The slides were then incubated with fitch‐secondary antibodies (1:200, Sigma‐Aldrich) in PBS 1× for 1 h in the dark, counterstained with DAPI (1 μg/mL; Sigma‐ Aldrich) diluted in PBS 1× for 5 min in the dark at room temperature and finally mounted in Vectashield (D.B.A. Italia). The number of positive cells was calculated as described by Lee et al. (2009): briefly, 12 different areas (1 mm²) randomly selected from each section were taken, and the number of signals was determined using ImagePro 3 software (NIH, Bethesda, US). The results were expressed as means ± SD (%).