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 due to its higher affinity, forming the dye-lipid complex. This result in red coloration of lipid droplets.

Discard the excess dye and observe the cells under a microscope. The intracellular lipid droplets appear intensely red.

Put a cell culture coverslip in every well in a 24-well plate, and seed HepG2 cells, as described in the text manuscript. After the appropriate incubation, wash the cells with 1 milliliter of PBS, and discard the supernatant. Fix them with 1 milliliter of 4% paraformaldehyde in PBS, and incubate for 1 hour at room temperature. Discard the excess paraformaldehyde, and rinse the cells with 1 milliliter of distilled water.

Then, add 1 milliliter of 70% isopropanol, and incubate for 5 minutes. Discard the excess isopropanol, and add 1 milliliter of Oil-red O solution, and incubate for 30 minutes. Discard the excess Oil-red O solution, and then, rinse with 1 milliliter of distilled water. Observe the cells under the microscope at a magnification of 400x.