Oil red o saturated solution

After induction of adipogenic differentiation, 3T3-L1 cells were rinsed three times with PBS and then fixed for 20 min with 4% PFA. The cells were treated with 60% isopropanol in H₂O for 2 min and then stained in freshly diluted Oil Red O solution (Oil Red O Saturated Solution (Solarbio, #G1260) was diluted with water (3:2), filtered through a 0.45 µm filter) for 30 min. Cells were then washed with 60% isopropanol in H₂O and twice with PBS. The cells were observed in H₂O under a microscope and photographed. Oil Red O was extracted with 100% isopropanol, and the absorbance reading was performed at OD₄₉₂ nm.

Oil Red O staining was performed using Oil Red O Saturated Solution (Solarbio Life Sciences, Beijing, China) according to the manufacturer’s instructions. Briefly, an Oil Red O working solution was prepared by mixing 3 parts of Oil Red O Saturated Solution with 2 parts deionized water. The transfected CRC cells were inoculated on 14 mm round coverslips in 24-well plates, washed twice with PBS after 24 h, and fixed in 4% paraformaldehyde for 30 min before staining. For frozen tissue sectioning, optimal cutting temperature (OCT)-embedded tissue was cut into 8 μm sections, which were fixed in 4% paraformaldehyde for 30 min before staining. The cells or tissue sections were incubated in 60% isopropanol for 30 s and then in Oil Red O working solution for 20 min at room temperature. Then sections were subsequently washed with 60% isopropanol and deionized water again, counterstained with haematoxylin (Solarbio Life Sciences, Beijing, China), and photographed by an Olympus microscope (Tokyo, Japan). The Oil Red O staining area was calculated as Oil Red O⁺ area divided by the area of hematoxylin staining and normalized to the average of the control.