An alkaline phosphatase (AP) assay was performed to visualize the ectopic sprout formation of the sub-intestinal venous plexus (SIVP) in zebrafish embryos, as according to Serbedzija and colleagues [66 (link)]. The AP assay was performed as described elsewhere [24 (link)]. Briefly, at 72 hpf, the control and treated embryos were fixed in 4% paraformaldehyde (Sigma-Aldrich), put into 100% methanol, equilibrated in Tris buffer (comprising 100 mM Tris-HCl pH 9.5, 50 mM MgCl₂, 100 mM NaCl, and 0.1% Tween-20), and stained with nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) solution (Blue staining solution, Merk KGaA, Darmstadt, Germany). The images of the stained SIVP were taken in a lateral position at 32X magnification with a Zeiss Axiozoom V13 (Carl Zeiss AG) microscope, equipped with a PlanNeoFluar Z 1×/0.25 FWD 56 mm lens and Zen Pro software (Carl Zeiss AG). The number of SIVP branches was manually counted in all experimental groups according to Goi and Childs [67 (link)], and as previously described [68 (link)], and was then graphed using GraphPad Prism 8 (GraphPad Software, Boston, MA, USA).
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