Gb22301

Briefly, VICs grown on 24-wells plates were fixed with 4% paraformaldehyde for 30 min at room temperature. Permeabilization was achieved by 0.5% Triton X-100, and VICs were then blocked for 0.5 h at room temperature using BSA (Maixin Biotech, Fujian, China). Primary antibodies against Vimentin (Santa Cruz, sc-6260, USA), α-SMA (Affinity, AF1032, USA), and CD31 (Abcam, ab28364, USA) were incubated overnight at 4°C. Subsequently, VICs were incubated with corresponding secondary antibodies labeled with fluorescein isothiocyanate (FITC; Servicebio, GB22301, Wuhan, China) for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Servicebio, GB22301, Wuhan, China) for 5 min. Fluorescent images were acquired using an inverted fluorescent microscope.

To observe the cell morphology, raw264.7 macrophages were cultured with 20 μg/mL Da-g-Xan for 4 h and then washed with PBS three times. Then, cells were fixed with 4% (w/v) paraformaldehyde for 30 min at room temperature, followed by 0.05% (w/v) Triton X-100 for permeabilization. Cytoplasm was stained with β-actin (abcam, ab115777) followed by donkey anti-rabbit antibodies (abcam, ab150075), and nuclei were stained with DAPI (abcam, ab104139). To co-localize CD206 with Da-g-Xan, raw264.7 macrophages were cultured with 20 μg/mL of Da-g-Xan-FITC for 4 h followed by staining with CD206 (abcam, ab195192). The nuclei were also stained with DAPI (abcam, ab104139). A confocal scanning microscope (FV1000, Olympus, Japan) was used for image analysis. The colonic anastomosis samples were fixed with 4% (w/v) paraformaldehyde, embedded in Paraffin, and then mounted on slides. The slides were treated with CD 31 (Servicebio, GB13063) and α-SMA (Servicebio, GB13044) followed by staining with donkey anti-goat antibodies (abcam, ab150135) and goat anti-mouse antibodies (Servicebio, GB22301). The images were recorded by an inverted fluorescence microscope (AXIO, ZEISS, Germany).