Ab24479

Sections from formalin-fixed, paraffin-embedded tumor tissues (4-µm thick) were immunostained and assayed using a PV6000 polymer system (ZSGB-BIO, Beijing, China). All paraffin sections were heated to 90°C for 20 min. Following routine deparaffinization by serial baths in xylene 3 times and dehydration in a graded series of ethanol (100, 95 and 80%) and distilled water, each section was treated with 0.3% hydrogen peroxide to block endogenous peroxidase activity. Sections were subsequently washed in 0.01 M phosphate-buffered saline (PBS; pH 7.4), microwaved at 100°C for 20 min for antigen retrieval and then incubated overnight at 4°C with optimal dilutions of primary antibodies. Antibodies used were anti-COX-2 rabbit polyclonal antibody (dilution, 1:400; ab15191; Abcam, Cambridge, UK) and anti-survivin rabbit polyclonal antibody (dilution, 1:1,200; ab24479; Abcam). Following washing in PBS, the tissue sections were treated with secondary antibody, IgG-horseradish peroxidase polymer multimer (PV-6000; ZSGB-BIO, Beijing, China), at 37°C for 20 min. Finally, the tissue sections were incubated with diaminobenzidine and counterstained with hematoxylin, cleared and mounted on neutralbalsam. In order to confirm COX-2 and survivin immunospecificity, 0.01 M PBS was supplied instead of primary antibody, as the negative control.