2.4. Histological Staining and NAFLD Activity Score (NAS)

For hematoxylin and eosin (H&E) staining, the tissues were first fixed in a 10% neutral buffered formalin solution. Then, they were embedded in paraffin, cut into 5 μm-thick sections, and stained with hematoxylin and eosin. For Oil Red O staining, the liver tissue was fixed in an optimal cutting temperature compound (Sakura, Torrance, CA, USA), cut into 10 μm-thick sections, and then stained with Oil Red O and Mayer’s hematoxylin solution for microscopy [24]. The stained sections were observed and photographed using a confocal microscope (Nikon Intensilight C-HGFI, Tokyo, Japan).

Steatosis, inflammation, and ballooning were graded according to the NAS criteria [25]. NAS includes the punctuation of steatosis, inflammation, ballooning, and fibrosis in routinely stained liver sections. All images were analyzed with ImageJ software (NIH, Bethesda, MD, USA).