Goat anti hif2α

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).

Formalin-fixed, paraffin-embedded lung sections were deparaffinized with xylene and progressively rehydrated in various concentrations of ethanol. Antigen retrieval was achieved with a citrate-based unmasking solution in a pressurized chamber (Decloaking Chamber, Biocare Medical, Pacheco, CA) by heating to 120 ^oC. Slides were washed with PBST, phosphate-buffered saline containing 0.5% Triton-X, for 10 minutes x3, and blocked for 30 minutes at room temperature in PBST solution containing 1% bovine-serum albumin. Incubation with primary antibodies, goat anti-HIF2α (R&D Systems, Minneapolis, MN), rat anti-VEGF_120/164 (R&D Systems, Minneapolis, MN), rabbit anti-Aquaporin-5 (Abcam, Cambridge, MA), rabbit anti-Vimentin (R&D Systems, Minneapolis, MN), and rabbit anti-SP-C (Abcam, Cambridge, MA), was performed overnight at 4 ^oC. After washing with PBST for 30 minutes x3, slides were incubated in secondary antibodies, Alexa Fluor-conjugated donkey anti-rat (Abcam, Cambridge, MA), anti-rabbit, and anti-goat (Invitrogen, Carlsbad, CA) IgG antibodies. Slides were washed again with PBST, dried, and mounted.