Paraffin-embedded liver sections were stained for F4/80 (anti-active macrophage) (Abcam, Cambridge, United Kingdom) and α-SMA (fibrosis hallmark) with immunohistochemistry (IHC) staining procedures as previously detailed[14 (link)]. Briefly, liver sections were incubated with a specific primary antibody, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibody (Dako, Glostrup, Denmark) and 3,3’-diaminobenzidine; the sections were then scanned with the NanoZoomer Digital Pathology system. Image-Pro Plus software was used to count F4/80+ cells and quantitatively analyze the staining intensity of α-SMA as previously described[13 (link)]. Six fields of view were randomly selected in each section.
Likewise, paraffin-embedded colon sections were stained for Zonula occludens-1 (ZO-1) (intestinal barrier hallmark) (Proteintech, Rosemont, IL, United States) with standard immunofluorescence staining procedures as previously detailed[15 (link)]. Briefly, sections were incubated with the rabbit polyclonal ZO-1 antibody, followed by incubation with Texas Red-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, United States) and 4’,6-diamino-2-phenyl indole (DAPI), and images were captured using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
Likewise, paraffin-embedded colon sections were stained for Zonula occludens-1 (ZO-1) (intestinal barrier hallmark) (Proteintech, Rosemont, IL, United States) with standard immunofluorescence staining procedures as previously detailed[15 (link)]. Briefly, sections were incubated with the rabbit polyclonal ZO-1 antibody, followed by incubation with Texas Red-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, United States) and 4’,6-diamino-2-phenyl indole (DAPI), and images were captured using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
