Rat anti mouse cd102 icam2

Rat anti-endomucin (Santa Cruz, sc-65495), goat anti-GFP FITC (Abcam, ab6662), rabbit anti-DsRed (Clontech 632496), rat anti-mouse CD102/ICAM2 (BD Biosciences, 553326), mouse anti-Twist1 (Santa Cru z, sc-81417), rat anti-mouse CD31/PECAM-1 (BD pharmingen 553370), rabbit anti-Sox17 (Zhou et al., 2015), mouse anti-smooth muscle actin (Sigma-Aldrich; C6198), rabbit anti-Runx2 (Abeam abl92256), rabbit cleaved caspase-3 (Cell Signaling 9661) and goat anti-EphB4 (R&D systems AF446). Alex Fluor-labeled secondary antibodies were from Invitrogen. See STAR Methods chart.

Primary mouse lung EC isolation was performed as described previously.^{26 (link)} Briefly, minced lungs were digested for 1 hour at 37°C in 0.1% collagenase IV (Sigma), passed through a 70-μm pore size cell strainer (BD Falcon) and cell suspensions were plated onto tissue culture plates coated with 0.1% gelatin, 10 mg/mL human plasma fibronectin (Sigma), and 30 mg/mL bovine collagen (Nutacon). One negative and 2 consecutive positive cell sorting steps were performed using Dynabeads M-450 sheep anti-rat IgG (Invitrogen) and purified rat anti-mouse CD16/CD32 Fcγ receptor, rat anti-mouse CD102 (ICAM-2, catalog no 553326, 1:200), and rat anti-mouse CD31 (PECAM1, clone MEC 13.3, 553370, 1:500; all from BD Pharmingen). Cells were grown on mouse lung EC media, consisting of 1 part of DMEM and 1 part of Ham’s F12 medium, including 0.1 μg/mL heparin (Sigma), 100 μg/mL penicillin/streptomycin (Gibco/Invitrogen), 6 mmol/L l-glutamine, 20% heat inactivated Fetal Calf Serum (Biowest), and 50 μg/mL endothelial mitogens (Biogenesis/Serotec). The media was supplemented with 500 nmol/L 4-hydroxytamoxifen (Sigma) to induce Cre-mediated gene deletion in culture. EC purity and inducible Cre-mediated loss of gene expression, after 48 to 72 h of induction were evaluated by immunofluorescence.

Antibodies used in this study were as follows: chicken anti-beta-galactosidase (Abcam 9361); rabbit anti-GLUT-1 (Thermo Scientific RB-9052-P0); rabbit anti-NG2 (Millipore AB5320); rat anti-mouse CD102/ICAM2 (BD Biosciences 553326); rat anti-PLVAP/ MECA-32 (BD Biosciences 553849); mouse anti-Claudin-5, Alexa 488 conjugate (Invitrogen 352588), and Texas Red streptavidin (Vector Laboratories SA-5006). AlexFluor-labeled secondary antibodies and GS lectin (Isolectin GS-IB4) were from Invitrogen. Primary antibodies were used at 1:200 to 1:500 dilution.