DNA fragmentation was detected using the In Situ Cell Death Detection Kit, TMR red (Cat. No. 12156792910 Roche, Sigma, St. Louis, United States) according to the manufacturer’s protocol42,43. Embryos at 72 hpf were treated with solution, and alloys were fixed overnight at 4 °C in a 4% paraformaldehyde solution. Fixed embryos were washed three times with PBS and then were incubated in a permeabilization solution for 1 hour at room temperature. After washing with PBS, embryos were treated with TUNEL solution mixtures for 60 minutes at room temperature in the dark and then washed with PBS 3 times before optical observation.
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