Visualizing ROS and Apoptosis in Zebrafish Embryos

Reactive oxygen species (ROS) in embryos were evaluated with dihydroethidium (DHE) fluorescent staining as in a previously described method [22 (link)]. In brief, zebrafish embryos (n = 20) at 5 h post-treatment were stained with DHE (30 μM). After 30 min incubation in the dark, embryos were rinsed twice with 1 × PBS and visualized under a fluorescent microscope (Nikon Eclipse TE2000, Tokyo, Japan) at the excitation and emission wavelengths of 588 nm and 605 nm, respectively. The apoptosis in the embryos was examined by acridine orange (AO) fluorescent staining [22 (link)]. At 5 h post-treatment, zebrafish embryos (n = 20) from different groups were suspended in 500 μL of AO (5 μg/mL). After 1 h staining, embryos were rinsed two times with 1 × PBS and visualized under a fluorescent microscope (Nikon Eclipse TE2000, Tokyo, Japan) at the excitation and emission wavelength of 502 nm and 525 nm, respectively. Image J software was employed for quantifying the fluorescently stained area.