Triglyceride reagents

Liver tissues were fixed in 10% neutral-buffered formalin for 48 h, embedded in paraffin wax and sectioned at 5 μm. For histological examination, sections were stained with hematoxylin and eosin and visualized using light microscope Olympus BX51 (Tokyo, Japan) equipped with Olympus DP22 digital camera (Tokyo, Japan). For the determination of hepatic lipid and TG levels, liver tissue was homogenized in extraction solution (chloroform: methanol = 2:1). Homogenates were mixed with 50mM NaCl and incubated at 4°C overnight, then centrifuged at 1,300g for 10 min at room temperature. The lower phase of extraction solution containing lipids was dried with nitrogen gas and weighed to determine the total lipid contents. The dried lipid pellet was dissolved in 1% triton X-100/PBS and used for TG content measurement using Triglyceride Reagents (Asan Pharmaceutical Co.).

HepG2 cells or rat hepatocytes were washed with phosphate-buffered saline (PBS) twice, fixed with 10% neutral-buffered formalin for 1 h, and then stained with oil red O solution for 1 h at room temperature. The stained lipid droplets in cells were examined under the microscope (Olympus CKX41, Tokyo, Japan) and photographed using an Olympus DP70 digital camera (Tokyo, Japan). Cellular oil red O was eluted with 100% isopropanol and quantified spectrophotometrically at 540 nm. For determination of intracellular triglyceride (TG) content, GSO-treated HepG2 cells or rat hepatocytes were washed twice with PBS. Cells were then scraped and centrifuged at 300g for 5 min. Cell pellets were lysed in 1% triton X-100/PBS. The concentration of intracellular TG was determined using Triglyceride Reagents (Asan Pharmaceutical Co., Seoul, Korea) according to the manufacturer's instruction, and normalized with protein concentration.