Lipid staining with Oil Red O

To evidence the lipid droplets accumulated in agLDL-exposed macrophages, Oil Red O staining was performed. The cells plated on coverslips placed in 24 well plates (5x10⁵ cells/well) were incubated for 24 h with agLDL, oxLDL or nLDL (100 μg protein/ml). After washing with phosphate buffer saline (PBS), the cells were fixed in 4% paraformaldehyde (10 min), washed twice with PBS, stained with Oil Red O (0.3% in 60% isopropanol, 15 min) to evidence the neutral lipids and examined by light microscopy with a Microphot FXA Nikon microscope using filter block B-2A.