Histological Evaluation of Liver Injury and Fibrosis

Liver samples were fixed with neutral-buffered formalin, embedded in paraffin and cut into 4 μm thick sections that were stained with Hematoxylin and eosin and Sirius red^{22 (link)}. F4/80-positive macrophages were detected immunohistochemically using a rat monoclonal anti-mouse F4/80 antibody (MCA497GA, Serotec, UK). Apoptotic cells were detected by TUNEL assay using an Apop-Tag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA), and anti-Caspase-3 antibody (ab13847, Abcam, Cambridge, UK). Sirius red-positive area was measured using the software WinROOF (Mitani, Chiba, Japan). TUNEL-positive cells and hCLS were counted in the whole area of each section and expressed as the mean number/mm². According to the NASH clinical research network scoring system, each score for steatosis, inflammation, and hepatocyte ballooning was evaluated by two investigators^{49 (link)}. Briefly, the degree of steatosis, inflammation, and hepatocyte ballooning was scored using hematoxylin & eosin-stained liver sections. Each variable was graded from zero to three, and the sum of the scores was considered as NAFLD activity score. Fibrosis was staged from zero to three using Sirius red-stained sections.