Anti type 1 collagen col1a1

Biochemical and histological analyses were performed as reported previously[19 (link)]. Plasma analytes included alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG) and total cholesterol (TC). Liver homogenates were analyzed for TG and TC. Paraformaldehyde-fixed liver pre- and post-biopsies were paraffin-embedded, sectioned, and stained with hematoxylin-eosin (Dako, Glostrup, Denmark), Picro-Sirius red (Sigma-Aldrich, Broendby, Denmark), anti-type I collagen (Col1a1; Southern Biotech, Birmingham, AL), or anti-galectin-3 (Biolegend, San Diego, CA, United States). The NAFLD activity score (NAS) and fibrosis staging system was applied to liver pre-biopies and terminal samples (drug treatment experiments) or only terminal samples (disease progression experiment) for scoring of steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis outlined by Kleiner et al[31 (link)]. All histological assessments were performed by a pathologist blind to treatment. Because all treatment paradigms affected total liver weight, quantitative data on liver biochemistry (liver TG, TC) and histology (liver lipid, galectin-3, Col1a1) were expressed as whole-liver amounts by multiplying individual terminal liver weight with the corresponding liver lipid concentration (biochemistry data) or percent fractional area (histology data), respectively.

Biopsy and terminal liver samples (both from the left lateral lobe) were fixed overnight in 4% paraformaldehyde. Liver tissue was paraffin-embedded and sectioned (3 µm thickness). Sections were stained with hematoxylin-eosin (HE, Dako, Glostrup, Denmark), Picro-Sirius red (Sigma-Aldrich, Broendby, Denmark), anti-galectin-3 (cat. 125402, Biolegend, San Diego, CA, United States), or anti-type I collagen (Col1a1; cat. 1310-01, Southern Biotech, Birmingham, AL, United States) using standard procedures[22 (link),23 (link)]. The NAS and fibrosis staging system was applied to liver pre-biopsies and terminal samples for scoring of steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis outlined by Kleiner et al[10 (link)]. Quantitative histomorphometry was analyzed using digital imaging software (VIS Software, Visiopharm, Hørsholm, Denmark)[22 (link),23 (link)]. Proportional (fractional) areas of liver fat (HE-staining), galectin-3 and Col1a1 were expressed relative to total sectional area. All histological assessments were performed by histologists blinded to the experimental groups.