Oil Red O Staining Procedure

Macrophages were gently washed with PBS, fixed with 4% phosphate buffered formalin for 15 min at 37°C and rinsed with PBS. The cells were then treated with 60% isopropanol for 15 s and stained in the darkness with Oil Red O solution for 30 min at 37°C. Oil Red O solution was freshly prepared by dilution of the stock solution (60 mg Oil Red O dissolved in 20 mL of 100% isopropanol) with distilled water in the ratio 3:2 and subsequent filtration. After washing with PBS the cells were counterstained with hematoxylin for 2 min, rinsed with PBS and analyzed under Nikon Eclipse Ti (Nikon, USA) microscope.