Histopathological Evaluation of Intestinal Inflammation

Upon euthanasia, distal colon segments were immediately postfixed in 10% formalin. The tissue sample was embedded in paraffin and stained with hematoxylin and eosin. The degree of enteritis, which uses a scoring system as previously described (Chen et al., 2008 (link)), consists of epithelial cell damage (score: 0–3), congestion/edema (score: 0–3), and neutrophil infiltration (score: 0–3). Two independent pathology professors double-blindly evaluated these sections. Alcian blue and periodic acid-Schiff (AB-PAS) staining were performed with the instructions (Solarbio). The sections were stained with p-mTOR, beclin1, zonula occludens-1 (ZO-1), occludin, and claudin-1 antibodies as previously described (Chung et al., 2014 (link)). Images were managed using the Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).