Ab114977

Paraffin‐embedded tissue sections were deparaffinised in xylene and hydrated with graded ethanol. After being treated with 3% hydrogen peroxide, the sections were blocked with 5% BSA and incubated with primary antibodies against HIF‐1α (1:50, ab114977, Abcam), METTL3 (1:50, ab195352, Abcam) and GPX4 (1:100, ab125066, Abcam) at 4°C overnight. Then, a secondary antibody conjugated with horseradish peroxidase was used, followed by diaminobenzidine staining and counterstaining with haematoxylin. The slides were analysed under light microscopy (Carl Zeiss).

Extracted protein samples were boiled for 5 min and separated by electrophoresis (Bio-Rad, Richmond, CA) in 12% SDS-PAGE gel before electroblotted (Bio-Rad) onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA) [11 (link)]. After polyvinylidene fluoride membranes were blotted with Tris buffer containing 5% fat-free dry milk and 0.05% Tween-20 (TBST; 0.05% Tween 20, 100 mmol/L of Tris-HCl, and 150 mmol/L of NaCl, pH 7.5) for 1 h at 25°C, they were rinsed in TBST for 4 times and incubated overnight at 4°C with primary antibodies, which were mouse polyclonal GAPDH (Abcom, ab22556), rabbit polyclonal HIF-1α (Abcam, ab114977), mouse monoclonal VEGF (Abcam, ab1316) and rabbit polyclonal VEGFR1 (Abcam, ab2350) with 1:3000, 1:2000, 1:2000 and 1:2000 dilutions, respectively. The membranes were washed in the same manner as described above and incubated with the horseradish peroxidase-conjugated secondary antibody (1:5,000; Sigma) for 40 min. Then, the membranes were washed 6 times for 5 min in TBST and the blots were detected using Lumi-light Western Blotting substrates (Biofuture, Beijing, China) and analyzed with the Imaging Analysis Software of National Institutes of Health (Bethesda, MD).

Tissue samples homogenization, protein extraction, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and WB was performed as previously described [27 (link)]. The membranes were subjected to blocking by using Tris buffered saline (TBS: 10 mM Tris-HCl, pH 7.4, 165 mM NaCl) added with 0.1% Tween 20 (TTBS) and 5% non-fat dry milk, at room temperature for 1 h. The anti-HIF antibody (#ab114977, Abcam) at 1:1000 dilution was incubated overnight at 4 °C.
Following four washing steps of 10 min in TTBS, donkey anti-rabbit secondary antibody conjugated with peroxidase was employed at 1:2000 dilution for 1 h at room temperature. After additional washing steps, bound antibody was visualized by enzyme chemiluminescence with Clarity ™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Milano, Italy). The blots were stripped and reprobed for β-actin (CP01, Calbiochem, San Diego, CA, USA) (1:500) as a loading control in order to perform normalization. Protein quantization and normalization were performed as reported in a previous study [27 (link)].