Zebrafish Intersegmental Vessel (ISV) Angiogenesis Assay

Transgenic zebrafish with vascular-specific fluorescence Tg (Flk-1:GFP) were used, and all experiments were conducted according to the standard ethical guidelines and under control of Biology Institute, Qilu University of Technology of committee, and were in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. After fertilized eggs were disinfected and washed, they were transferred to zebrafish embryo culture water at 28°C with controlled light. After fertilized eggs developed for 20 h post-fertilization (hpf), normal developing zebrafish larvae were selected, placed in 24-well plates (8 zebrafish larvae/well) and treated as follows: blank control group (embryo culture water), model group of PTK787 (Sigma, 0.2 μg/ml), curcumin (Sigma, 50 μM curcumin + 0.2 μg/ml PTK787) and Cur20 group (50 μM Cur20 + 0.2 μg/ml PTK787). After the drugs were incubated for 24 h, (i.e. 44 hpf), the effect of drugs on ISVs length of zebrafish was observed and calculated under a fluorescence microscope with Image Pro Plus software (Chen et al., 2018).