4.5. In Vivo Subintestinal Angiogenesis Assay on Zebrafish Embryos

Occurring at 48 hpf, transgenic embryos were treated for 24 h with different concentrations of vandetanib (VAN) and cabozantinib (CAB). Stock solutions of VAN and CAB, prepared in dimethyl sulfoxide (DMSO), were diluted in fish water to concentrations ranging from 5 × 10⁻⁷ up to 1 × 10⁻⁵ M, and from 5 × 10⁻⁸ up to 5 × 10⁻⁶ M for VAN and CAB, respectively. As a control, some embryos were incubated with a fish medium containing the same concentration of DMSO used for the drug treated embryos. Occurring at 72 hpf, sub-intestinal vein (SIV) plexus images were taken with a Leica M205 FA stereomicroscope equipped with a Leica DFC 450 C digital camera using the LAS software (Leica Microsystems, Wetzlar, Germany). Vertical vessels that composed the SIV basket were counted in the controls and in the treated embryos to analyze physiological angiogenesis.