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

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.