Rabbit anti hif1α

Cells were lysed with 10 mmol/L Tris at pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% SDS, and protease/phosphatase inhibitor cocktail (#78440, Thermo Fisher). Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, blotted with primary antibodies overnight at 4 °C, and detected using horseradish peroxidase-conjugated secondary antibodies followed by exposure to chemiluminescence reagents (#PI34580, Thermo Fisher). The following antibodies were used: rabbit anti-HIF1α (#10006421, 1:500, Cayman), goat anti-HIF2α (#AF2997, 1 µg/ml, R&D Systems), mouse anti-beta actin (#MA1-91399, 1:50,000, Invitrogen), HRP-linked anti-rabbit IgG (#7074, 1:10,000, Cell Signaling), HRP-linked anti-mouse IgG (#7076, 1:60,000, Cell Signaling), and HRP-linked anti-goat IgG (#705035147, 1:20,000, Jackson ImmunoResearch).

We used paraformaldehyde-fixed, paraffin-embedded samples of one microgram tissue thickness as previously described [36 (link)]. In essence, heat-induced epitope retrieval was performed in a citrate buffer at pH 6.0 followed by diverse blocking steps and detection with the ABC Vectastain kit (Maravai Life Sciences, San Diego, USA). Immunohistochemistry for HIF-1α was done with the Dako CSAII-Kit (Dako Agilent, Santa Clara, USA). As primary antibodies we used rabbit-anti-HIF-1α (Cayman Chemical, Ann Arbor, USA Cat# 10006421, RRID:AB_409037) at a dilution of 1:10.000, rat-anti-F4/80 (Bio-Rad, Hercules, USA Cat# MCA497, RRID:AB_2098196) at a dilution of 1:200, rat-anti-VEGF (BioLegend, San Diego, USA Cat# 512901, RRID:AB_2212504) at a dilution of 1:300, rabbit-anti-PHD2 (novus Biologicals, Cat# 100–137, RRID:AB_350074 at a dilution of 1:200. For the detection of F4/80 and VEGF, biotinylated anti-rat antibody (Santa Cruz Biotechnology, Dallas, USA Cat# sc-2041, RRID:AB_631752) and for the detection of PHD2, biotinylated anti-rat antibody (Santa Cruz Biotechnology, Dallas, USA, Cat# sc-2040, RRID:AB_631743) was used as the secondary antibody (1:200).