Immunohistochemical Analysis of Key Markers

IHC analysis of Ki-67, claudin, and lipopolysaccharide (LPS) were performed on formalin-fixed, paraffin-embedded tissues. Four μm-thick sections were deparaffinized, rehydrated, and rinsed. For antigen retrieval, the sections were microwaved in Vector Unmasking Solution (Vector Laboratories Burlingame, CA, USA) and treated with 3% hydrogen peroxide. Sections were incubated with 200 μL of a primary antibody at a 1:100 dilution. The following primary antibodies were used: Ki-67 (mouse monoclonal antibody, clone MIB-1, Dako), claudin-3 (rabbit polyclonal antibody, RB-9251-P1, Thermo Fisher), LPS (mouse monoclonal antibody, clone WN1 222-5, Hycult Biotech, USA). Secondary antibodies and avidin/biotin complex used were from the Vector Vectastain ABC Elite Kits (VectorLabs, Burlingame, CA, USA). For visualization, sections were treated with DAB (Dako), counterstained with hematoxylin, dehydrated, and mounted in a xylene-based mounting media. Images were taken on a Nikon Ni-U microscope. Quantification was performed using the FIJI image software, as described^{33 (link)}, with at least 20 images per sample for intestinal tissues and liver, and at least 9 images for sLN (magnification x400). Positive signal was isolated via color threshold; percent area positive for the marker was measured for each image and the mean was calculated.