Fluoview fv1000 confocal laser scanning microscope system

Embryos were fixed with 4% formaldehyde (Polysciences) for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Aldrich) for 30 min, and then blocked with blocking solution (10% Fetal Bovine Serum (Hyclone), 0.1% Triton X-100) for 1 hr at room temperature, or overnight at 4°C. Primary Antibodies used were: mouse anti-CDX2 (Biogenex, CDX2-88), goat anti-SOX2 (Neuromics, GT15098), rabbit anti-PARD6B (Santa Cruz, sc-67393), rabbit anti-PARD6B (Novus Biologicals, NBP1-87337), mouse anti-PKCζ (Santa Cruz Biotechnology, sc-17781), rat anti-CDH1 (Sigma Aldrich, U3254), mouse anti-ZO1 (Thermo Fisher Scientific, 33–9100), mouse anti-YAP (Santa Cruz Biotechnology, sc101199), rabbit anti phospho-YAP (Cell Signaling Technologies, 4911), chicken anti-GFP (Aves, GFP-1020). Stains used were: Phallodin-633 (Invitrogen), DRAQ5 (Cell Signaling Technologies) and DAPI (Sigma Aldrich). Secondary antibodies conjugated to DyLight 488, Cy3 or Alexa Flour 647 fluorophores were obtained from Jackson ImmunoResearch. Embryos were imaged using an Olympus FluoView FV1000 Confocal Laser Scanning Microscope system with 20x UPlanFLN objective (0.5 NA) and 5x digital zoom. For each embryo, z-stacks were collected, with 5 µm intervals between optical sections. All embryos were imaged prior to knowledge of their genotypes.

HUVECs were seeded at 1 × 10⁴ cells/well onto coverslips in a 12-well plate until 60% confluent. Serum-starved cells were treated with or without 100 μM CaD/25 ng/mL VEGF for 15 min (for actin filaments detection) or 2 hours (for ZO-1 expression). The cells were then fixed in 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 (for ZO-1 staining the cells were not permeabilized). Actin filaments were stained by phalloidin-Alexa fluor 488 (1:250) for 1 h at room temperature and nuclei were detected by DAPI. Images were captured by Leica DM 1400B confocal microscope. For ZO-1 detection, the cells were stained with ZO-1 antibody (1:100) and subsequently with Alexa fluor 488 secondary antibody (1:250). The slides were examined with Olympus Fluoview FV1000 confocal laser scanning microscope system (Olympus, Tokyo, Japan). The concentration of VEGF and pVEGFR-2-Tyr1175 in mouse kidney lysates was measured using commercially available ELISA kits.