Bx53 biological microscope

Histopathological analysis was performed by incising standardized specimen from specified portions of liver. Briefly, liver tissues were fixed in 10% formalin for 4 hours, and dehydrated in a graded series of ethanol and embedded in paraffin wax. 4-μm-thick sections were cut and stained with hematoxylin-eosin (H&E) staining and analyzed under Olympus-BX53 biological microscope. To evaluate fat deposition, fresh liver samples were embedded using tissue OCT-freeze medium and sliced into 8–10 μm-thick sections using a cryostat microtome (LEICA CM1850). The frozen sections were then stained with Oil red O for 15 minutes before being analyzed under an Olympus-BX53 biological microscope.

HepG2 cells were cultured for 24 hours and induced with FFA (OA/PA) and/or CG mono-compounds (CG-3, CG-4, CG-10, CG-14). The concentration for positive drug was 20 μM and the concentrations for mono-compounds were at 5, 10 and 20 μM. TG was measured by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and proteins were normalized in each group after BCA. Nile red is a selective fluorescent stain for intracellular lipid droplets. Cells, fixed with 4% buffered paraformaldehyde for 15 min, were washed with1 mL PBS and then stained with Nile red (Sigma-Aldrich); Stock solution (0.5 mg/mL in DMSO) was diluted 1:5000 in PBS; 500 μL of this diluted Nile red solution was added into each well (six well plate) and protected from light at room temperature for 5 min and then washed with 1 mL PBS. Pictures were taken with the Olympus-BX53 biological microscope. For the quantitative analysis, HepG2 cells treated with different group of drugs were harvested, and incubated with Nile red (1 μg/ml) in dark for 5 min at room temperature. The fluorescence intensity of each sample was measured immediately by flow cytometry at an excitation wavelength of 488 nm and emission wavelength of 550 nm.