Oil red o solution

Frozen liver tissues embedded in FSC 22 frozen section compound (Leica, USA) were sectioned at 8 mm, mounted on silicone coated slides (Leica, USA), and then stained with oil red O solution (Cayman chemical, USA) for 10 min at 60°C and Mayer’s hematoxylin solution for 1 min. All of the slides were observed under an Olympus BX61 microscope (Tokyo, Japan) and photographed using an Olympus DP70 digital camera (Tokyo, Japan).

Liver tissues fixed in 10% neutral buffered formaldehyde were sliced (5 μm in thickness) and trimmed transversely into serial sections using a cryo-microtome (LEICA Microsystem, Wetzlar, Germany). Sections were subsequently deparaffinized in xylene, rehydrated through an ascending ethanol series and stained with haematoxylin-eosin (HE) for 1 min and then with oil red O solution (Cayman Chemical, Ann Arbor, MI, USA) for 15 min at 60°C. Mounting was performed with xylene-based media. The slides were observed under an Olympus BX 61 microscope (Olympus Tokyo, Japan) at 200X magnification and images were acquired using Olympus DP70 digital camera (Olympus).

3T3-L1 cells (3×10⁴/well) were seeded on 96-well plates and adipocyte differentiation was induced for 6 days. Cells were washed with phosphate-buffered saline (PBS), fixed with 10% formalin for 30 min, and then washed with 60% isopropanol. After air drying, 0.3% Oil-red O solution (CAYMAN CHEMICAL COMPANY. Ann Arbor, MI, USA) was added to each well at room temperature for 30 min. After the solution was removed, cells were washed with distilled water and air dried. Oil-red O in triglyceride droplets was extracted with 100% isopropanol and determined at OD₅₁₀, as previously described, for quantification [20 (link)].