2.15. Zebrafish Subintestinal Vessel (SIV) Formation

The Tg (fli:EGFP) zebrafish embryos were collected, and embryos with the same development stage (around 2.5 hpf) were selected. Groups containing at least 25 embryos were treated with the indicated compounds at 0.5 µM for 3 days. The egg water was refreshed every day, and the abnormal embryos were removed. After 3 days of treatment, the zebrafish embryos were fixed in 4% PFA for 2 h at room temperature and stored at 4 °C in PBS. The area containing SIV in each zebrafish embryo was scanned with an Andor Dragonfly 500 spinning disk confocal by using a 488 nm laser. At least ten zebrafish embryos in each group were randomly selected and analyzed. The SIV vasculature of the zebrafish was quantified with ImageJ. The leading buds were counted as previously described [39]. The experiments were repeated twice with similar results.