NO and ROS staining in zebrafish larvae

The production of NO and ROS in zebrafish larvae was visualized using 4-amino-5-methylamino-2'7'-difluorofluorescein diacetate (DAF-FM-DA, Sigma-Aldrich Chemical Co.) and DCF-DA, respectively, 24 h after chemical treatment as previously described (Jeong et al., 2018[19]). In brief, zebrafish embryos (4 dpf) were transferred to 24-well plates and incubated with 5 µM DAF-FM-DA and 20 µM DCF-DA for 30 min and visualized using the CELENA^® S Digital Imaging System (Logos Biosystems, Anyang, Gyeonggido, Republic of Korea). Fluorescence intensities were calculated using ImageJ software (Wayne Rasband, National Institute of Health, Bethesda, MD, USA) and expressed as a percentage compared to the untreated control.