Huc d

For the myenteric plexus, fresh gastric tissues were placed in pre-cooled phosphate-buffered saline (PBS) solution. Thereafter, the mucosa and muscularis tissues were separated and fixed in 4% paraformaldehyde for 10 min. The paraffin-embedded gastric tissue sections were dewaxed and hydrated and subjected to antigen retrieval. The tissues were incubated with donkey serum containing 0.3% Triton X‐100 at 4 °C overnight for blocking of nonspecific binding. Subsequently, the tissue sections were incubated overnight at 4 °C with the specific primary antibodies: GFAP (ABclonal, Wuhan, China), HuC/D (Abcam, Cambridge, UK), S100B (Abcam, Cambridge, UK), β-Tubulin (ABclonal, Wuhan, China) H3K9me1 (A2358, ABclonal), H3K9me3 (A2360, ABclonal), H3K27me1 (A2361, ABclonal), and H3K27me3 (A2363, ABclonal). After washing three times with PBS, the preparations were then stained with the secondary antibody and incubated for 2 h at room temperature. The cell nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) for 20 min. The specimens were observed using a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Immunofluorescence assay was performed based on the methods described previously [32 ]. Briefly, the intestine tissues were quickly dissected on ice, and subjected to fixation in fresh prepared 4% paraformaldehyde. The frozen sections were prepared and washed with phosphate-buffered saline. After incubation with blocking solution for 1 h, the sections were further incubated with primary antibodies against TuJ1 (1:250, purchased from Abcam Co., Cambridge, UK) and HuC/D (1:250, obtained from Abcam Co., Cambridge, UK) at 4 °C overnight. On the next day, the sections were subsequently incubated with the fluorescent dye-conjugated secondary antibodies at room temperature for 1 h. Then, the nuclear of cell was visualized by 4’,6-Diamidino-2-phenylindole (DAPI). Lastly, the sections were observed under a fluorescence microscope.