4 6 diamidino 2 phenylindole staining solution c1005

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was used to determine the jejunal apoptosis. The whole experiment was performed using a commercial TUNEL BrightRed Apoptosis Detection Kit (A113, Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions. First, the jejunal sections were deparaffinized, rehydrated, then incubated with Proteinase K (20 mg/ml) at room temperature for 20 min. Second, the sections were incubated with the terminal deoxynucleotidyl transferase enzyme with BrightRed Labeling Mix at 37°C for 60 min in the dark. Finally, the sections were stained with 4′,6-diamidino-2-phenylindole staining solution (C1005, Beyotime Biotechnology, Shanghai, China) for 5 min in the dark. To ensure there was no nonspecific reaction, the negative control was performed without incubation of the TdT enzyme. The fluorescent images were acquired using a LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

BMSCs at third passage were seeded in cell dish (801002, NEST). After the cells reached 80% confluence, BMSCs were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 20min. Then the cells were blocked with bovine serum albumin (BSA) for 1 h. Subsequently, the cells were incubated with primary antibody against AhR (NB300-515, 1:100, Novus) at 4 °C overnight. After washing, cells were incubated with dylight 594-conjugated secondary antibody (1:200, A23420, Abbkine) for 1 h at room temperature. Then, the cells were stained with 4',6-diamidino-2-phenylindole staining solution (C1005, Beyotime) for 5 min. Finally, the stained cells were observed and photographed under confocal microscope (Leica-LCS-SP8-STED).