Establishment of a visual model for monitoring neutrophil migration in aanat2 −/− zebrafish. (A) Hybridization of transgenic line Tg(lyz:EGFP) labeling neutrophils and aanat2 mutants for two consecutive generations. (B) In F2 zebrafish, we identified the homozygous aanat2 −/− transgenic zebrafish. Extracted genomic DNA from the tail fin of screened transgenic zebrafish was amplified using PCR and digested using the MspI enzyme. The uncleaved band indicated homozygous zebrafish. (C) To evaluate the functional effect of Tg(lyz:EGFP);aanat2 −/− zebrafish, we measured melatonin levels using an ELISA. Fifty larvae were homogenated and then melatonin was extracted using methanol as an individual sample (IBL international, Germany). The experiment used three samples. Results indicated that melatonin was significantly decreased at night compared with WT/AB larvae. The melatonin content could be partly rescued by injection of aanat2 capped mRNA (control, n = 50; mutant, n = 50; mutant + mRNA, n = 50) (ANOVA analysis). (***P < 0.001).