Rabbit anti sp c

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.

The following reagents were used for immunofluorescence: rat anti-platelet endothelial cell adhesion molecule 1 (PECAM1; BD Pharmingen, 553370, 1:250-1000), mouse anti-α-smooth muscle actin-Cy3 (Sigma-Aldrich, C6198, 1:100-1000), rat anti-E-cadherin (Invitrogen, 53-3249-82, 1:500), rabbit anti-phosphohistone H3 (EMD Millipore, 06-570, 1:250) mouse anti-HOPX (E-1; Santa Cruz Biotechnology, sc-398703, 1:100), rabbit anti-SPC (Abcam, ab90716, 1:500). Prolong Gold mounting media (±DAPI) and Alexa Fluor fluorescent secondary antibodies (Alexa 488 donkey anti-rat A21208, Alexa 594 donkey anti-rabbit A21207, both diluted to 1:1000) were purchased from Invitrogen. The nuclear stain DRAQ5 (62251, 1:5000) was purchased from Thermo Fisher Scientific. Antibody titrations were performed on WT tissues to determine the optimal dilution for each tissue. Cells and explants were cultured in the following reagents: interferon-γ (R&D Systems, 485-MI-100), Dulbecco's modified Eagle's medium (DMEM; Corning Life Sciences, 10-013-CV), fetal bovine serum (FBS; Thermo Fisher Scientific), and penicillin-streptomycin (Thermo Fisher Scientific, 15140122).