Embryos were placed 5 per well in 48-well plates containing 400 µl of embryo medium/0.1% DMSO. Test compounds were added directly to the embryo medium. For intersegmental vessel (ISV) angiogenesis assay, embryos were treated from 6–24 hpf and from 2–5 dpf for hyaloid vessel (HV) angiogenesis assay [17] (link). Larvae were left to incubate with drugs at 28°C on a 14 h light/10 h dark cycle, euthanized and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 2 hours before analysis. Drugs were purchased from the following: Rapamycin (Selleckchem S1039), NVP-BEZ235 (Selleckchem S1009), PI-103 (Selleckchem S1038), LY294002 (Sigma L9908), A66 (Selleckchem S2636), WYE125132 (Selleckchem S2661), Palomid 529 (Selleckchem S2238), XL147 (Selleckchem S1118) and XL765 (Selleckchem S1523). 2–5 dpf zebrafish larvae were euthanized in ice-cold PBS, or 4% PFA, or lysis buffer.
Free full text: Click here