Ki 67

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.

Cells or tissue specimens were homogenized in RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA), separating lysates by electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gel. The various proteins were then electrophoretically transferred onto polyvinylidene fluoride membranes for blocking (5% skim milk, 1 h) and overnight incubation (4 °C) with the following primary antibodies: K1 (Abcam), K10 (Abcam), K14 (Abcam), Ki-67 (Acris Antibodies), p-AKT (Cell Signaling Technology, Danvers, MA, USA), AKT (Cell Signaling Technology), involucrin, and β-actin (Santa Cruz Biotechnology). Immunoreactive bands were developed using a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology) and visualized as directed by light emission (Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate; PerkinElmer, Waltham, MA, USA). Quantitative assessment of immunoreactivity was software-enabled (IMT Inc).