Occludin and zo 1

Liver histology was assessed using the NAFLD activity score (NAS), as described previously [24 (link)]. Staining and counting of the number of neutrophilic granulocytes and assessment of hepatic fibrosis were carried out as described previously [25 (link)]. Liver sections were stained for F4/80, iNOS and 4-hydroxynonenal protein adducts (4-HNE) using polyclonal antibodies (F4/80: Abcam, Cambridge, UK; iNOS: Affinity BioReagents, Rockford, USA; 4-HNE: AG Scientific, San Diego, USA) and staining was evaluated as described before [6 (link)]. Paraffin-embedded sections of proximal small intestine (4 µm) were stained and analysed for the tight junction proteins occludin, zonula occludens 1 (ZO-1) and hydroxyindole-O-methyltransferase (HIOMT), respectively, using polyclonal primary antibodies (occludin and ZO-1: Invitrogen, CA, USA; HIOMT: Biozol Diagnostica GmbH, Germany) as previously described [19 (link)].

Paraffin-embedded intestinal tissue sections (4 μm) were stained for 3-NT, occludin and ZO-1 as well as Arg-1 and -2 as previously described [21 (link),26 (link),27 (link)]. In brief, sections were incubated with specific primary antibodies (3-NT: Santa Cruz Biotechnology, USA; occludin and ZO-1: Invitrogen, USA; arginase-1 and arginase-2: Cell Signaling, USA) following peroxidase-linked secondary antibody and diamino-benzidine to determine specific binding. The extent of occludin and ZO-1 staining was defined as the percentage of microscopic field within the default color range capturing eight pictures per sample using a microscope with an integrated camera (Leica DM6B, Leica DMC4500, Leica, Germany). 3-NT-positive cells were counted per mm villus in eight randomly selected microscopic fields per sample.

Immunohistochemical staining methods were used to measure concentration of 4-HNE, iNOS and MyD88 in paraffin embedded liver sections as well as of MMP13, Occludin and ZO-1 in small intestinal tissue sections as previously described^{7 (link),19 (link),22 (link)}. In brief, after treating sections with citrate buffer (MMP13) or incubating with protease (occludin, ZO-1) and blocking tissue sections with bovine serum albumin solution (iNOS, MMP13, MyD88), they were incubated with specific primary antibodies (4-HNE: AG Scientific, San Diego, CA, USA; iNOS: Thermo Fisher Scientific, Waltham, MA, USA; MMP13: LifeSpan BioSciences, Seattle, WA, USA; MyD88: Santa Cruz Biotechnology, Dallas, TX, USA; Occludin and ZO-1: Invitrogen, Carlsbad, CA, USA). Subsequently, sections were incubated with peroxidase-linked secondary antibodies followed by diaminobenzidine (Peroxidase Envision Kit, DAKO, Hamburg, Germany). Staining was evaluated using a camera integrated in a microscope (Leica DM4000 B LED, Leica, Wetzlar, Germany) and an analysis system (Leica Applications Suite, Leica, Wetzlar, Germany) as described previously^{19 (link),42 (link)}.