Mouse anti zo 1

Embryos for Phalloidin staining were fixed for 30 min in 4% paraformaldehyde (PFA), washed 3 times for 5 min with 0.8% TritonX in PBS (PBT) and incubated for 2 hr at room temperature with Phalloidin (Invitrogen, 1:40 in 2% PBT). For immunohistochemistry embryos were fixed in 4%PFA at 4°C for the following antibodies: c-Myc (9E10) Santa Cruz Biotechnology (sc-40) (1:500), Anti-Acetylated Tubulin (T679) Sigma-Aldrich (1:500), mouse anti-ZO1 (Zymed; 61–7300) (1:200). Glyo-Fixx (Thermo Scientific, UK) at 4°C was used as a fixative for these antibodies: Di-pERK1/2 (Diphosphorylated ERK-1 and 2 antibody-mouse (M8159) Sigma-Aldrich) (1:500), Anti – ROCK – 2a (CT), Z – FISH- rabbit (AS-55431) (1:50), Mouse Anti-E-Cadherin (610182) BD-Biosciences (1:500), and Bouin’s fixative (Polysciences) was used for Phospho-Myosin Light Chain 2 (Ser19) Antibody (#3671 Cell Signaling (1:20)).

Whole-mount and tissue section immunohistochemistry was performed as previously described^{12 (link),16 (link)}. In this study we used mouse anti-ZO-1 (339111; Zymed) at 1:300, anti-rabbit Phospho-Myosin Light Chain 2 (Ser19) (3671; Cell Signaling) at 1:20 in 2.5% normal goat serum (Sigma). For secondary antibodies we used anti-mouse Alexa 488 and 633 (Molecular Probes) at 1:1,000 in 2.5% normal goat serum. For F-actin labelling we used Phalloidin (A12379; Invitrogen) at 1:1,000 in 2.5% normal goat serum. For analysis of neural tube organsition tissue sections were made every 14 mm on a HM 560 MV Micron microtome.

Mutant and wild-type siblings were anesthetized and washed for 1.5 min in PBS with .3% Triton X-100 for permeabilization, then fixed with 2% formaldehyde in 0.1 M PIPES, 1.0 mM MgSO₄ and 2 mM EGTA overnight at 4 °C. The embryos were manually deyolked under a dissecting microscope and blocked for 1 hour in PBS with 4% BSA and 0.3% Triton X-100. Primary and secondary antibodies were incubated overnight. Washes were done after each step with 0.3% Triton X-100 in PBS for 3 h to remove excess antibodies. Antibodies used include mouse monoclonal anti-Nkx6.1 (1:20; Developmental Studies Hybridoma Bank (DSHB), Iowa), rabbit polyclonal anti-Prox1 (1:200; Millipore), goat polyclonal anti-Hnf4a (1:50, Santa Cruz), rabbit polyclonal anti-pan-Cadherin (1:5000; Sigma), mouse monoclonal anti-Islet1/2 (1:20; DSHB), mouse monoclonal 2F11 anti-Anxa4^{41 (link), 70 (link)} (1:1000; gift from J. Lewis, Cancer Research UK), mouse monoclonal anti-Alcama (1:20; DSHB), mouse anti-ZO1 (1:200, Zymed), chick or rabbit polyclonal anti-GFP (1:300; Torrey Pines Biolabs), rabbit anti-cleaved Caspase-3 (1:50; Cell Signaling). Imaging was done with a Zeiss 710 confocal microscope with Zen9 software and images were processed with Adobe Photoshop CS3.