Histology
Paraffin-embedded liver samples were cut into approximately 4 μm sections and stained with haematoxylin and eosin (HE) and Sirius red. For Sirius red staining, deparaffinized sections were incubated for 15 min with Picrosirius red solution prepared by mixing 5 mL of 1% Sirius red solution (Muto Pure Chem. Co., Ltd., Tokyo, Japan) with 100 mL of saturated picric acid solution (Fujifilm Wako Pure Chem. Corp). The NAFLD activity score (NAS) of each slide was evaluated as previously described (Kleiner et al., 2005).