Ab83178

hOPCs were prepared in the Paediatric Laboratory of the Sixth Medical Centre of PLA General Hospital, China, using previously established methods of cultivation and identification. Briefly, hOPCs were induced by NSCs. The cells were cultured in a self-made medium at 37 °C in a humidified 5% CO₂ incubator (Additional file 1: Text S1). The hOPCs were identified using immunofluorescence staining. Monoclonal mouse anti-PDGFR-α (1:250, Cat. #C2318, CST, Boston, MA, USA), rabbit anti-NG2 (1:50, Cat. #ab83178, Abcam, Cambridge, Cambridgeshire, UK) and mouse anti-A2B5 (1:50, Cat. #MAB1416, R&D Systems, Minneapolis, MN, USA) antibodies were used to identify hOPCs. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:20, Cat.#28718-90-3, Sigma-Aldrich, St. Louis, MO, USA) for 5 min and then observed using fluorescence microscopy (IX-70, Olympus Corporation, Tokyo, Japan).

Colons were removed from mice, Swiss-rolled, and submerged in 4% paraformaldehyde overnight at 4°C. The following day, colons were washed five times and placed in a 15% sucrose-PBS solution overnight, then moved into a 30% sucrose-PBS solution for 24 h. The Swiss-rolled colons were embedded in optimal cutting temperature compound (OCT) and sectioned (20-μm thickness). The colon sections were blocked (PBS containing 10% goat serum, 5% BSA, and 0.5% triton X-100) and incubated overnight at room temperature with an Alexa Fluor 488 labeled rat monoclonal antibody against E-cadherin (Santa Cruz Biotechnology; sc-59778 AF488) and rabbit antibodies against CSPG4 (Abcam; ab83178). After 3 washes, the sections were incubated with a Cy3 labeled goat anti-rabbit antibody (Abcam; ab6939) for 1 h at room temperature, washed, and mounted in Prolong Gold with DAPI. Stained sections were imaged on a Nikon W1-CSU/SoRa super-resolution confocal microscope using a 10X objective. For each channel, an equivalent amount of background was subtracted using Nikon NIS Elements v4.2. Brightness and contrast were applied equally for all images using ImageJ v2.1.0.

Cells were seeded in duplicates into µ-Dish 35 mm (60 000 cells/ibidi dish). After 48 h, cells were washed with sterile phosphate-buffered saline (PBS, Thermo Fisher) before being fixed in 4% paraformaldehyde for 30 min (10 min at 4°C followed by 20 min at room temperature). Cells were washed and then permeabilized for 4 min in 0.2% Triton in PBS. Cells were washed again and incubated in blocking buffer (1% bovine serum albumin, and 2% normal goat serum, in PBS) for 1 h at room temperature before incubating with the primary antibody for 2 h. The following primary antibodies were used: Nestin (Abcam, ab22035, 1:100), Vimentin (Abcam, ab8069, 1:200), NG2 (Abcam, ab83178, 1:200), Ki67 (Abcam, ab16667, 1:200), Vinculin (FAK100, Sigma 1:200), and F-actin (FAK100, Sigma 1:500). Secondary antibodies were applied for 1 h (goat anti-mouse Alexa Fluor 488 preadsorbed, Abcam, 1:750 dilution and goat anti-rabbit Alex Fluor 594 preadsorbed, Abcam, 1:750). Nuclei were stained with DAPI (Roche).