Zeb1 antibody

The streptavidin–biotin–peroxidase immunohistochemical staining method was used in this study. Four-micrometer sections of formalin-fixed paraffin-embedded tissue were mounted on poly-L-lysine-coated slides. The slides were deparaffinized, and then endogenous peroxidase activity was blocked with 3% hydrogen peroxide in 50% methanol for 10 min at room temperature. The slides were rehydrated and washed with phosphate-buffered saline (PBS) and then pretreated with citrate buffer (0.01 M citric acid, pH 6.0) for 20 min at 100°C in a microwave oven. After nonspecific binding sites were blocked by incubating in 2% normal goat serum in PBS for 15 min at 37°C, the slides were incubated overnight at 4°C with the primary rabbit ZEB-1 antibody (dilution 1:100; Abcam) and rabbit E-cadherin antibody (dilution 1:200; Abcam). The slides were washed with PBS and incubated with biotin-labeled secondary antibody (dilution 1:100; Zhongshan Biotechnology) for 1 hour at 37°C and then incubated with avidin–biotin-conjugated peroxidase and diaminobenzidine (Zhongshan Biotechnology), and counterstained with hematoxylin. Slides were dehydrated with different concentrations of alcohol and soaked in xylene, and then mounted with neutral balsam and visualized using light microscope.