Rat anti cdh1

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.