Mouse anti p53 monoclonal antibody

After being deparaffinized and pretreated in citrate buffer, sections were incubated with normal goat serum for 15 min at room temperature. Sections were applied with PBS overnight at 4°C, with mouse anti P53 monoclonal antibody (Thermo Scientific, MS-187), rabbit anti CCNE1 polyclonal antibody (Sigma-Aldrich, HPA018169), mouse anti CDKN2A monoclonal antibody (Santa Cruz Biotechnology, sc-56330). The secondary antibody, biotinylated anti-rabbit and mouse immunoglobulin, was applied for 15 min at room temperature and sections were rinsed in PBS. Peroxidase conjugated streptavidin was applied for 15 min, followed by Diaminobenzidine (DAB) substrate for 10 min and hematoxylin for5 min. Finally, sections were rinsed with water, dehydrated, cleared and mounted with permanent mounting medium. Immunohistochemical staining was analyzed using Image Pro-Plus (version 6.0; Media Cybernetics, Silver Spring). Briefly, the positive staining area was selected as the area of interest (AOI). The area sum and integrated optical density (IOD) of the AOI were selected as the measurement parameters. The target protein expression was analyzed by comparing the IOD (P53, CCNE1and CDKN2A) in different groups. Finally, statistical analysis of the mean expression index for each duplicate was performed.