Sta 612

Hepatic fat accumulation was determined by Oil Red O staining. Briefly, liver cryosections (5 μm thick) were fixed with 4% PFA in PBS for 20 min and incubated in freshly prepared Oil Red O working solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for 10 min and counterstained with hematoxylin for 15 sec. The random images of each Oil Red O stained liver frozen tissue sections (n = 3 per group) were captured at ×200 magnification. Relative areas of steatosis, expressed as % of fat accumulation, were quantified by histomorphometry using WinROOF image analyzer (Version 7, Mitani Corp, Tokyo, Japan). Moreover, total hepatic lipid was extracted from fresh frozen liver tissues using a lipid extraction kit (STA-612; Cell biolabs, San Diego, CA, USA). Hepatic triglycerides and cholesterol were then quantitated using colorimetric assay kits, according to the manufacturer’s instructions (Fujifilm Wako).