Hematoxylin and eosin (H&E) and oil red O staining

For H&E staining, the liver tissue was fixed with 10% formaldehyde for 24 h at 4°C, embedded in paraffin and cut into 10 mm sections according to a standard protocol. The sections were stained with H&E (hematoxylin, 10 min; eosin, 30 sec; room temperature). The adipose tissue or frozen sections of liver were washed with phosphate buffered saline twice, fixed with 10% formalin at room temperature for 10 min and then stained with oil red O (Sigma-Aldrich; Merck KGaA) at 60°C for 10 min. Subsequently, images were captured using a light microscope (magnification, ×200 or ×400; Nikon Corporation). Oil red O stained sections were evaluated using Image-ProPlus 6.0 software (Media Cybernetics, Inc.) and H&E staining sections were evaluated according to The Pathology Committee of the NASH Clinical Research Network's histological feature scoring system (35).