Liver Histology and Immunohistochemistry Protocol

Liver histology (H&E staining) and the NAFLD activity score were assessed as previously described.[26] (link), [27] , [28] Sirius Red or Trichrome staining, provided by a Pathology Core at the University of Kentucky, was used to analyze liver fibrosis. For liver immunohistochemical staining, liver paraffin sections were stained with anti-CD41 antibody (Thermo Fisher Scientific; MA, USA), anti-neutrophil antibody [7/4] (ab53457) (Abcam; Cambridge CB20AX, UK), anti-p-selectin antibody (Novus Biologicals, CO, USA), anti-CD4 antibody (Abcam; Cambridge CB20AX, UK), or anti-CD8 antibody (Novus Biologicals, CO, USA), followed by incubation with biotinylated secondary antibody, peroxidase substrate diaminobenzidine (Vector Lab) and counterstaining with hematoxylin. Images were captured by a Nikon Eclipse 55i microscope. Semi-quantification of neutrophil, CD4, or CD8 positive cells in liver sections was performed as previously described.²⁹ (link)