Goat anti ephb4

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.

Embryos harvested from timed matings were fixed by immersion and lungs from postnatal mice were inflation fixed using 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Following overnight immersion in 4% PFA/PBS, fixed tissue was processed according to standard protocols for paraffin or frozen embedding. Hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence were performed on tissue sections (5–10 μm) as previously described (Lange et al., 2009 (link)). Primary antibodies included guinea pig anti-Sox17 (Seven Hills Bioreagents), goat anti-endomucin (R&D Systems), rat anti-Pecam-1 (BD Pharmingen), GSL-IB4-biotin (Vector Labs), rat anti-CD34 (Abcam), goat anti-EphB4 (R&D Systems), and mouse anti-alpha smooth muscle actin (Sigma). Fluorophore-conjugated secondary antibodies included Alexa Fluor-488 and Alexa Fluor-594 (Jackson ImmunoResearch and Life Technologies). For fluorescent stains, sections were stained with DAPI and mounted with ProLong Gold anti-fade reagent following antibody labeling (Invitrogen). Bright-field images were obtained using a Zeiss Axio ImagerA2 microscope equipped with AxioVision Software. Fluorescent images were obtained using a Nikon A1Rsi inverted laser confocal microscope and analyzed using Imaris software (Bitplane Scientific Software).