Immunostaining of Zebrafish Embryos

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.