Histochemical detection of esterified cholesterol within human atherosclerotic lesions using the fluorescent probe filipin

The fluorescent cholesterol probe filipin has been used in this study to histochemically examine the morphology and cholesterol composition of intra- and extracellular Sudanophilic lipid deposits which accumulate in human atherosclerotic lesions. Because filipin reacts with unesterified but not esterified cholesterol, detection of cholesteryl esters was carried out by first extracting native unesterified cholesterol with ethanol, and then enzymatically converting esterified to unesterified cholesterol before filipin staining. The size and structure of particles comprising extracellular cholesteryl ester-rich lipid deposits was different in regions of necrosis as detected using filipin compared with the Sudan lipid-soluble dye oil red 0. Whereas oil red 0 staining often indicated that extracellular cholesteryl ester in these regions occurred in amorphous and spherical particles of varying sizes, filipin staining revealed that extracellular cholesteryl ester occurred in spherical particles more uniform in size than indicated by oil red 0 staining. Also, the majority of extracellular cholesteryl ester-rich particles in necrotic regions were smaller than intracellular cholesteryl ester-containing lipid droplets. In addition, the use of filipin to detect intracellular cholesteryl ester allowed distinction of 2 subpopulations of oil red 0-stained cells which did and did not contain detectable cholesteryl ester.