Histological Evaluation of Liver Fibrosis and Steatosis

Liver sections from formalin-fixed paraffin-embedded tissue were stained with haematoxylin and eosin (H&E) or picrosirius red to evaluate fibrosis. Fibrosis quantification was performed using Image J (NIH). Optimal cutting temparature compound (OCT)-embedded frozen liver sections were stained with Oil Red O to assess steatosis. For immunohistochemistry, sections were incubated overnight at 4°C with anti-CD68 (Cell Signaling Technology, 97778S), anti-MPO (Abcam, ab9545) or anti-4 hydroxynonenal (4HNE, Abcam, ab46545) primary antibodies. Antibody detection was performed using SignalStain Boost IHC Detection reagent (HRP rabbit, Cell Signaling Technology, 8114S) and immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) as a substrate (Cell Signaling Technology, 8059S). Nuclear counterstain was performed using haematoxylin. Stained sections were imaged with a 10X objective using a Nikon Ni-U microscope. CD68-and MPO-positive cells were quantified using QuPath (Centre for Cancer Research and Cell Biology, Belfast, UK^{17 (link)}).