Pa5 114687

The expression of activated Caspase-3 and P53 proteins was examined using the immunocytochemistry technique. In brief, cell suspensions were cultured on sterile gelatin slides. After 24 h, the slides were washed with PBS and fixed for 20 min with 4% paraformaldehyde at 4 ˚C. Subsequently, the slides were washed again with PBS and incubated for 20 min in 2N HCl, followed by exposure to Triton X-100 for 30 min to permeabilize the cells. To block non-specific antigen sites, the slides were then incubated with 10% Goat serum for 30 min. Next, the cells were incubated overnight at 4 ˚C with primary antibodies against P53 (PA5-27822, Invitrogen) or active Caspase-3 (PA5-114687, Invitrogen) appropriately diluted with PBS (1:100). After thorough washing with PBS, the cells were exposed to secondary FITC-conjugated anti-rabbit antibodies (1:200) (31635, Invitrogen) for 1 h at room temperature in the dark. Following another round of PBS washing, DAPI was added to stain the nuclei, and the cells were examined with a fluorescent microscope (TCM 400 Binocular Microscope, Labomed). Cell counting was performed using Image j software, and the ratio of cells expressing P53 or caspase 3 to the total number of cells was reported as a percentage of P53 and caspase 3 expression.