β catenin

For immunohistochemical analysis, embryos at E9.5 were embedded in gelatin (15% sucrose and 7.5% gelatin in PBS) and cryo-sectioned at thicknesses of 10 μm and 50 μm on a Leica CM1950 cryostat. Sections of at least three different embryos per primary antibody were blocked with PBT (PBS, 10% Triton and 1% BSA) and incubated overnight at 4°C with the primary antibody against myosin heavy chain II-B (PRB-445P, Covance) diluted 1:150 in PBT; β-catenin (610153, BD Biosciences) diluted in PBT 1:200; ZO1 (40-2200, Invitrogen) diluted 1:100 in PBT; or Vangl2 (a kind gift from Mireille Montcouquiol, Neurocentre Magendie, Bordeaux, France) (Belotti et al., 2012 (link)) diluted 0.5:150 in PBS and 5% BSA; or Pax6 [mouse monoclonal, Developmental Studies Hybridoma Bank (DSHB)] diluted in PBT 1:200. The secondary antibodies used were as follows: goat anti-rabbit FITC-conjugated for the primary antibodies against myosin II, ZO1 and Vangl2 (ab6717, Abcam) or a Cy™3-conjugated affiniPure goat anti-mouse IgG to detect antibodies staining β-catenin and Pax6 (115-165-166, Jackson ImmunoResearch). Phalloidin-Tetramethylrhodamine B isothiocyanate was used to visualize F-actin (P1951, Sigma-Aldrich). Sections were mounted with Hydromount (HS-106, National Diagnostics), and then photographed by using an Olympus (Tokyo, Japan) BX61 microscope or a confocal microscope (TCS-SP2-AOBS, Leica).