Histological Analysis of NAFLD Progression

Paraffin blocks were sectioned into 4 μm slices and used for hematoxylin and eosin (HE) staining, Sirius red staining, and immunohistochemistry (IHC) assay according to standard protocols. Frozen liver tissue sections were stained with Oil Red O (G1260, Solarbio). For pathological grading, all liver specimens were scored by two experienced pathologists according to the NAFLD activity score (NAS), defined as the sum of steatosis (0–3), inflammation (0–3), and hepatocyte ballooning (0–2). An NAS score ≥5 was considered to indicate NASH. Samples were scanned using a slide scanner (Pannoramic DESK, 3D Histech kft, Hungary). For quantitative analysis, the areas of lipid droplets and Sirius red staining were quantified using ImageJ software (version 1.6.0; NIH, Bethesda, MD, USA). Immunohistochemical staining was semi-quantitatively analyzed using the immunoreactive scoring system^{73 (link)}. The percentage of positive cells was graded on a scale of 0−4: (0: negative, 1: 0–25%, 2: 26–50%, 3: 51–75%, 4: 76–100%). The signal intensity was scored on a scale of 0–3: 0 = negative; 1 = weak; 2 = moderate; and 3 = strong. Thus, the final immunoreactive score = (score of staining intensity) × (score of percentage of positive cells).