G1262

In situ cells were stained with oil red O staining solution (G1262, Solarbio Biotechnology, Beijing, China) to observe the accumulation of lipid droplets in cells. Briefly, we removed the cell culture, then washed it with PBS 2 times, and added Oro Fixative 20–30 min fixation solution. The fixed solution was removed and washed with distilled water two times. The cells were soaked in 60% isopropanol for 5 min. Then, isopropyl alcohol was removed and a newly prepared Oro Stain was added to soak for 10–20 min. The cells were washed with water 2–5 times until there was no excess dye. Mayer hematoxylin staining solution was added and the nuclei were restained for 1–2 min. After washing the cells again 2–5 times, ORO Buffer was added for 1 min. Finally, the cells were covered in distilled water and examined under a microscope.

Here, Oil Red O staining was performed to evaluate the adipogenic differentiation of BMSCs. After 16 days of inducing adipogenesis, the culture medium was aspirated, the cells were rinsed with PBS, fixed using a 4% paraformaldehyde solution, and stained with a pre-prepared Oil Red O working solution (G1262, Solarbio, China) at room temperature. After staining, the cells were washed with PBS to remove any excess staining solution. Lastly, each well was filled with PBS, and the plate was examined under a microscope (DSZ2000X, Cnmicro, China) to assess the effectiveness of adipogenic staining.

To analyze lipid deposition in adipocytes, Oil O staining (G1262, Solarbio, China) was performed for the different groups. To analyze lipid deposition in the human subepicardial layer, modified Oil O staining (G1261, Solarbio, China) was performed. Images were captured using a light microscope (ECLIPSE E100; Nikon, Tokyo, Japan).

ORO staining was performed to detect the accumulation of myelin debris in the LC after SCI and the accumulation of myelin debris in RAW 264.7 cells. For cell experiments, RAW 264.7 cells were treated with myelin debris or D-4F, and the concentration gradient settings of the two were based on previous studies [17 (link)]. The cells to be treated were first washed twice with PBS and fixed with an ORO fixative (G1262, Solarbio, China) for 20–30 min. The fixative was discarded, and the cells were washed twice with distilled water and stained with ORO staining solution (G1260, Solarbio, China) for 20 min. The staining solution was discarded, and the cells were washed 3 times with distilled water. Hematoxylin was added to counterstain the nucleus for 2 min. Finally, ORO buffer was added for 1 min, and the cover glass was sealed with distilled water. For tissue experiments, frozen sections of the spinal cord were dried and washed with PBS, stained with ORO staining solution for 10 min, treated with 60% isopropanol for 5 min, and finally washed with distilled water. Representative images were obtained by fluorescence microscopy.

BMSCs were cultured in adipogenesis induction medium (5 μg/mL insulin, 0.5 mmol/L 3‐isobutyl‐1‐methylxanthine, and 1 μmol/L dexamethasone) containing TAA, ORI or TAA + ORI for 14 days. BODIPY staining (GLPBIO, GC42959-5, 1:1000 dilution) and oil red O staining (Solarbio, G1262) were used to detect the formation of adipocytes.