Whole Mount Embryo Immunostaining Protocol

Embryos were harvested in 1X PBS and fixed in 4%PFA in PBS at 4°C overnight. Standard whole embryo immunostaining was performed as previously described (Inman et al., 2013 (link); Sandell et al., 2014 (link)). Primary antibodies against the following proteins were used: β-tubulin III (TuJ1) at 1:1000 dilution (Covance Research products, cat# MMS-435P), PECAM1/CD31 at 1:400 dilution (BD Pharminogen, cat# 553370), cleaved caspase 3 at 1:1000 dilution (Cell Signaling Technology, cat# 9661S) and Sox10 at 1:500 dilution (Abcam, cat# 155279). Secondary antibodies used were: Goat anti-Mouse IgM (Invitrogen/Molecular Probes, cat# A-21042), Goat anti-Rabbit IgM (Invitrogen/Molecular Probes, cat# A-11034) and Peroxidase AffiniPure Donkey Anti-Rat IgG (H + L) (Jackson ImmunoResearch, cat# 712-035-153) at 1:500 dilution. Immunostained embryos were mounted in Vectashield Antifade Mounting Media (Vectorlabs, cat# H-1000) and imaged using an upright confocal microscope. Except for Pecam1 staining in Figure 1, control and mutant embryos were imaged with the same magnification.