Immunohistochemical Analysis of Liver and Intestinal Markers

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)}.