Oil Red O Staining: A Technique for Staining Neutral Lipids in Hepatocytes for Detecting Hepatic Steatosis In Vitro

Hepatic steatosis is a pathological condition characterized by an excessive accumulation of fats in hepatocytes - a prominent liver cell type. The fats deposit in the form of lipid droplets of varying sizes in the cytoplasm of the hepatocytes.

Structurally, a lipid droplet contains a core of neutral, hydrophobic fats such as triglycerides and cholesterol esters. The core is surrounded by a phospholipid monolayer decorated with various proteins.

To visualize fat deposition, take a multi-well plate containing an adherent hepatocyte culture. The cells are pretreated with a steatogenic medium to induce lipid droplet accumulation inside them.

Add the desired concentration of paraformaldehyde, which diffuses into the cells, and cross-links the proteins. This step deactivates proteases and preserves the hepatocyte structure. Discard the paraformaldehyde and add the Oil-red-O solution containing the organic dye dissolved in isopropanol.

The isopropanol acts as a carrier solvent for the sparingly soluble Oil-red-O molecules. This helps the dye molecules to enter fixed hepatocytes and eventually reach the lipid droplets. Once inside, the dye transfers from the isopropanol to neutral lipids d