Pb0084

Primary antibodies against the following were used: HIF-1α (mouse monoclonal, 1:1 500, MAB5382; Chemicon Inc, Billerica, MD); BNIP3 (mouse monoclonal, 1:3000, B7931; Sigma, St. Louis, MI); BCL-xL (rabbit polyclonal, SAB3500349; 1:1000, Sigma, St. Louis, MI); VEGF (rabbit polyclonal, 1:500, PB0084; Boster, Wuhan, China); GAPDH (mouse monoclonal, 1:10000, KC-5G4; KangCheng, Shanghai, China); and β-tubulin (mouse monoclonal, 1:1000, A06868; Boster, Wuhan, China). Horseradish peroxidase-labeled secondary antibodies were obtained from Zymed Laboratories Inc (San Francisco, CA). Western blot assays were performed as previously described [16 (link)].

Tissues were fixed in 10% (v/v) formaldehyde, embedded in paraffin, and then 4 μm sections were cut and used for H&E staining with primary antibodies. The slides were treated with EDTA for antigen retrieval and incubated with endogenous peroxidase blocking solution to inhibit endogenous peroxidase. HRP-streptavidin conjugated secondary antibody (Vector Laboratories) was used and detected by the DAB kit (Vector Laboratories). Primary antibodies were visualized by slides scanner (KF-PRO-005, Ningbo, China). We used quantitative scoring methods as scores 0, 1+, 2+, and 3+, representing positive cells of none or rare, <25%, 25–75%, and >75%, respectively. According to the product datasheet, TR4 antibody (ab58365, Abcam) stains nuclei. HIF1a (ab51608, Abcam) antibody stains cytoplasm and nuclei (mainly), VEGFA antibody (PB0084, Boster) stains cytoplasm, and P63 antibody (PB0164, Boster) that is used as a quality control stains nuclei.