Determination of melanin contents in zebrafish

Melanin contents in zebrafish were determined following the previously reported method.34,35 For melanin contents determination, 50 µM dose was selected on the basis of in vivo depigmentation results. The collected synchronized embryos were arrayed by dropper into the dish: 20±2 embryos were used in the dish and treated with compound 6d and 50 µM of reference drug kojic acid in 3 mL of E3 medium. After 72 hpf, embryos were anesthetized in tricaine methanesulfonate MS-222 solution. After anesthetizing, embryos were washed three times with E3 medium and eyes were removed from all the treated and non-treated embryos. Embryos extract (pellet) was prepared by homogenization and centrifugation, and the pellet was dissolved in 1 mL of 1N NaOH at 100°C for 10 min. The level of melanin was measured at an absorbance of 405 nm, and the result was compared with a standard curve of synthetic melanin. All experiments were repeated three times.