Reactive oxygen species (ROS), superoxide anion, nitric oxide detection, and quantification of AP sites

ROS were detected by one of several dyes obtained from Invitrogen (Eugene OR, USA). General ROS were detected by CM-H₂DCFDA, superoxide anion was detected by MitosoxRed, and nitric oxide (NO) was detected by DAF-FM acetate. They are activated by interaction with ROS to which they are sensitive and only fluoresce upon exposure to light of the appropriate exciting wavelength. Dyes were added to fish water at a final concentration of 10 µM for CM-H₂DCFDA and DAF-FM acetate, or 1 µM for MitosoxRed. After embryos were washed three times in fish water to remove exogenous dye, they were examined by confocal fluorescence microscopy. Eight to ten embryos were examined in each group.

To quantify AP sites, genomic DNA was prepared from Apex1 knockdown embryos and controls using QIAamp DNA Mini Kit (Qiagen, Boston, MA, USA) in the presence of deferoxamine (0.1 mM) to prevent oxidation during DNA preparation⁶⁸. AP site assay was performed using the aldehyde reactive probe from Dojindo (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) according to the manufacturer’s instructions. Two hundred embryos were used for each group.