Tunel reagent

After fixation in 10% formalin, the intestinal tissues were embedded in paraffin wax and cut into 3 μm slices. The slices were then dehydrated with gradient alcohol, cleaned with xylene, and sealed with resin. Hematoxylin and eosin (H&E) staining was performed on the slices. Finally, images were captured using a light microscope (Nikon, Tokyo, Japan) to detect histopathological changes. The severity of intestinal injury was assessed according to Chiu’s scoring system, as previously described [9 (link)].
For immunohistochemistry (IHC), tissue slices were treated with anti-Fas antibody (1:500 dilution; Cell Signaling Technology). The horseradish peroxidase (HRP)-conjugated secondary antibody (Gene Tech, Shanghai, China) was incubated for 30 min at room temperature and used to detect the primary antibody. The images were acquired using a light microscope (Nikon), and Fas staining was quantified using Image-Pro Plus 7 (Media Cybernetics, MD, USA).
Apoptotic cells in the intestinal epithelium sections were detected using TdT-mediated dUTP-biotin nick end-labeling (TUNEL) reagent (Elabscience, Wuhan, China) according to the manufacturer’s instructions. Images were acquired using a fluorescence microscope (Leica Microsystems), and TUNEL-positive cells were quantified using Image-Pro Plus 7 (Media Cybernetics).

Cells were seeded in 6-well culture plates at 3 × 10⁵ cells/ml. Cells were fixed with 4% paraformaldehyde. 0.5% Triton X-100 was used to increase the permeability of the cell membrane. Cells were then co-incubated with the TUNEL reagent (Elabscience, China) for 1 h. Nuclei were stained with 4',6′-diamidino-2-phenylindole (DAPI) for 5 min. Images were observed on a fluorescence microscope.