Villin

Intestinal tissues of mice and minipigs were fixed with 10% neutral buffered formalin solution, embedded in paraffin wax, and sectioned transversely at a thickness of 4 µm. The slides were stained with H&E and Congo red. Histological scores were quantified in the H&E-stained slides and assessed by the degree of the epithelial maintenance, crypt damage, vascular dysfunction, and inflammation with infiltration of inflammatory cells. To investigate radiation-induced goblet cell damage, the slides were stained with PAS. For immunohistochemical analysis, the slides were subjected to antigen retrieval for 20 min and then treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min to block endogenous peroxidase activity. After washing with PBS, the slides were blocked with 10% normal goat serum (Vector ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) and incubated with primary antibodies, such as anti-Cldn3 (Invitrogen, Carlsbad, CA, USA), villin (Abcam, Cambridge, UK), Mpo (Abcam), ki-67 (Acris), Olfm4 (Invitrogen), and CD68 (Abcam) antibodies. Additionally, the slides were incubated with horseradish peroxidase-conjugated secondary antibody (Dako, Carpinteria, CA, USA) for 60 min. The peroxidase reaction was developed using diaminobenzidine substrate (Dako) prepared according to the manufacturer’s instructions, and the slides were counterstained with hematoxylin.