Liver histology

Sections of liver from the left lateral, right medial and caudal lobe were embedded in paraffin [37]. Sections of 3 µm thickness were then stained with Mayer’s Haematoxylin and Eosin (H&E, Sigma-Aldrich). Cryo-sections were stained with Oil Red O (Sigma-Aldrich) to evaluate and confirm the presence of hepatic steatosis. Presence of hepatocyte ballooning was evaluated on H&E stains. Hepatic inflammation was visualized using immunohistochemistry (IHC) to detect CD68-positive (CD68+), CD11b-positive (CD11b+) and CD45-positive (CD45+) cells. Details on the different IHC protocols are shown in Additional file 1: Table S1. For a small subset of animals, a double-staining of CD68 (IHC) and Oil Red O was performed on cryo-sections from the left lateral liver lobe, to characterize the content of CD68+ cells. Collagen deposition and hepatic stellate cell activation was evaluated using histochemical staining with Picro Sirius Red (Sigma-Aldrich) and IHC for α-SMA (Additional file 1: Table S1), respectively. All sections were scanned using a NanoZoomer 2.0 HT slide scanner (Hamamatsu, Hamamatsu City, Japan) and subsequently evaluated using NanoZoomer Digital Pathology Image Software (Hamamatsu). Image analysis for quantification of inflammation (CD68, CD11b, CD45), collagen deposition (Picro Sirius Red) and hepatic stellate cell activation (α-SMA) was performed with VIS software (Visiopharm, Hoersholm, Denmark). With automated threshold analysis applications, the area with positive staining for each marker of interest was identified in each tissue section and expressed as a percentage of the total area of the tissue section (i.e., the fractional area). For each marker of interest, the same threshold for identification of positive staining was used across all tissue sections in the study.