Sab3500340

The 4-µm thick slices were baked at 72°C for 1 h, and then dewaxed and rehydrated through xylene and alcohol with graded concentrations. Hydrogen peroxide (3%) was used to eliminate activity of endogenous peroxidase for 15 min. After PBS washing 3 times, the antigen retrieval was performed in a pressure cooker with EDTA (pH 8.0) (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). After cooling to room temperature, the sections were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with primary rabbit polyclonal anti-PTK7 antibody (1:7,500, SAB3500340; Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. The ready-to-use EnVision™ reagent (EnVision™ detection system peroxidase/DAB, rabbit/mouse; Dako, Glostrup, Denmark) was then used to bind the primary antibody. The 3,3′-diaminobenzidine tetrahydrochloride kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. was used to develop substrates. After being counterstained with hematoxylin, the sections were dehydrated with graded alcohol and xylene.