Zebrafish Husbandry and Embryo Collection

The zebrafish were kept in Zebrafish Drug Screening Platform of Shandong Academy of Science. Zebrafish of wild-type (AB line) and transgenic zebrafish lines [Tg(cmlc2:EGFP) (Huang et al., 2003), Tg(fli:EGFP) (Hu et al., 2012), Tg(l-fabp:EGFP) (Her et al., 2003), Tg(Lyz : EGFP) (Hall et al., 2007)] used in this study were feed according to standard procedures. Briefly, fish were kept with constant temperature at 28 ± 0.5°C and 14/10 hour light/dark photoperiod in an aquarium. The fish were fed with live brine shrimp twice a day and dry flakes once a day. The pH value of the water varies from 7.2 to 7.4, and the conductivity varies from 450 to 500 µS/cm. Before mating, adult male and female zebrafish were separated in a breeding tank at the ratio of 2:1. After the light on the next morning, embryos were generated by natural mating. Then the eggs were collected, rinsed three times and cultured in incubator before subsequent experiments. The embryos were checked at 6 hours post fertilization (hpf) under the microscope to make sure all of the embryos stay at the same development stage. At 24 hpf, embryos were examined under the microscope to make sure the lethal rate lower than 0.5%. Then the embryos can be used in the subsequent experiments. Two people will take part in one experiment, and one made the solution and treated the embryos, the other will take the picture and make the analysis.