Fat accumulation and lipid staining

Prior to Oil red O staining, embryos were submitted to a high fat diet composed of cooked egg yolk. Twenty wild type embryos at 5 hpf were placed in each well of a 6-well plate, containing 5 ml of medium solution E3. At 3 days post-fertilization, larvae were treated with different concentration of TSA and VPA. From 6 to 9 days post-fertilization, the larvae were fed each morning with 30 ml of high fat diet (2.4% egg yolk). At 10 days, larvae were fixed and neutral lipid accumulation was revealed with 0.3% Oil red O staining [24], during 4 hours. After washing, larvae were stored in 70% glycerol.