3.5. Zebrafish Embryo Imaging and Cardiotoxicity Assay

Imaging of zebrafish embryos was performed with Zeiss SteREO Discovery V8 Microscope equipped with Hamamatsu Orca Flash high-speed camera and HCImage software, version 4.4.1.0 (Hamamatsu Photonics, Tsukuba, Japan) as described in [64]. 96-hpf selected embryos were placed on a depression slide with 1–2 drops of 3% methylcellulose. Embryos were oriented and positioned on their side for measurement of the two major blood vessels, dorsal aorta (DA) and posterior cardinal vein (PCV). The captured images were analyzed using ImageJ software version 1.52a (NIH, Washington DC, USA) bundled with Java 1.8.0 in which variations in the size of the yolk, eye, and body length were measured. Moreover, videos of the tail were recorded for all embryos at the same site where the two major blood vessels were visible. Then, MicroZebraLab Blood Flow software from ViewPoint (Lyon, France) was used to measure three parameters; blood flow velocity, vessel diameter, and heart rate for both blood vessels [64].