Goat serum

Immunofluorescence analysis was performed according to standard protocols^{59 (link)}. Cells (1.5 × 10⁴) were seeded in triplicate on coverslips (WHB, Shanghai, China) in 24-well plates. After 48 h, the cells were fixed with 70% ethanol and permeabilized with 1.5 M HCl (Lanxi Zhongxing Chemical Reagent Co., Ltd., Lanxi, China). The cells were then washed twice with PBS and blocked with 5% goat serum (Absin Bioscience, Inc., Shanghai, China) and 0.3% Triton^TM X-100 (Sigma-Aldrich) in 1× PBS. Next, the cells were stained with primary antibodies against 5mC (#28692, 1: 1600, Cell Signaling Technology) or at 4 °C overnight^{34 (link)}. The next day, the cells were incubated with anti-rabbit IgG-Alexa Fluor^TM 488 antibody (#8878; 1:500, Cell Signaling Technology) in the dark on a gyratory shaker (Kylin-Bell Lab Instruments, Haimen, Jiangsu, China). Coverslips were then mounted with 5 μL of mounting medium containing DAPI (Beyotime). At least 100 cells from each biological replicate were captured at 600× magnification using a confocal laser-scanning microscope (Nikon Corporation). The fluorescence signal intensity was measured using ImageJ v. 2.4.1.7 (National Institutes of Health, MD, USA).

The formalin-fixed, paraffin-embedded intestinal pathological tissues were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min. After being washed with phosphate buffered saline (PBS, Solarbio, China), the sections were subjected to 15 min treatment with Triton X-100 (Solarbio, China) and 1 h blocking using 5% goat serum (Absin, China). Then, the sections were incubated with primary antibodies against (CX3CL1, 1:200 and iNOS, 1:200) for 24 h. The next day, the sections were cultured with secondary antibodies Alexa Fluor 488-Labeled Goat anti-Mouse IgG (1:1000, Beyotime, China) and Alexa Fluor 488-Labeled Goat anti-Rabbit IgG (1:1000, Beyotime, China), followed by DAPI staining (Beyotime, China). The results were observed under a confocal microscope.