Zen 3.1 zen pro software

Immunofluorescence (IF) microscopy was used to determine the nuclear translocation of ERK1/2 (MAPK). Cells were cultured on a sterile coverslip in 3.4 cm diameter culture plates. After irradiation and incubation, the cells were fixed (4% paraformaldehyde, 15 min at room temperature), washed (0.1% Tween-20 in PBS), permeabilised (0.1% Triton X-100 in PBS, 15 min at room temperature), washed, and blocked (1% BSA in PBS, 1 h at room temperature). Cells were exposed to unlabelled (1:250) primary antibody (Anti-MAP Kinase, activated (Dephosphorylated ERK-1&2) antibody, mouse monoclonal; M9692, Sigma-Aldrich, Johannesburg, South Africa) and incubated for 1 h at room temperature. The slides were then washed, and fluorescently labelled secondary antibody diluted in PBS-T (1:100) (anti-mouse, IgG, H&L, FITC conjugate; 12-506, Sigma-Aldrich, Johannesburg, South Africa) was added and incubated for 1 h at room temperature in the dark. Thereafter, the slides were washed and nuclei counterstained with 1 µg/mL 4′,6′-diamidino-2-phenelendole (DAPI), mounted, and observed under a fluorescent microscope, Carl Zeiss Axio Z1 (Carl Zeiss, Randburg, South Africa). Images were analysed by using ZEN 3.1 (ZEN pro) software (Carl Zeiss, Randburg, South Africa).