Whole-Mount in situ Hybridization Protocol

Antisense riboprobes were synthesized using T7 polymerase with a DIG RNA labeling kit (Roche) according to the manufacturer’s instructions. The ephrinb2, mrc1, flk, and stabilin probes have been described [22 (link),49 (link),50 (link)]. The whole-mount in situ hybridization protocol was based on Thisse et al. [51 (link)]. Briefly, embryos were fixed with 4% paraformaldehyde and stored at −20 °C in methanol. After rehydration, the riboprobes were added to hybridization at 65 °C overnight. After washing and blocking, an AP-conjugated anti-Dig antibody (Roche) was added and reacted with the NBT/BCIP substrate (Roche). The embryos were embedded in 3% methylcellulose and photographed.