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

Overview

In this video, we demonstrate in vitro staining of neutral lipids in hepatocytes to detect hepatic steatosis or excessive fat accumulation in liver. Excessive fat is accumulated in the cells in form of lipid droplets. Oil Red O is a lipid soluble dye that binds to intracellular neutral lipids in these lipid droplets. When observed under a light microscope, these intracellular lipid droplets appear intensely red in color.

Protocol

1. Lipid staining with Oil-Red O

1. Put a cell culture coverslip in every well in a 24-well plate.
2. Seed 100,000 HepG2 cells per well. Add 1 mL of standard RPMI 1640.
3. Pre-incubate at 37 °C and 5% CO2 for 24 h.
4. Change standard RPMI 1640 medium for the steatogenic medium.
5. Incubate for 24 h, 2 days, 3 days, and 4 days, refreshing the steatogenic medium every 24 h.
6. After the appropriate time, discard the supernatant.
7. Wash with 1 mL of phosphate buffered saline (PBS). Discard the supernatant.
8. Fix with 1 mL of 4% paraformaldehyde in PBS.
9. Incubate for 1 h at room temperature.
10. Discard the excess of paraformaldehyde.
11. Rinse the cells with 1 mL of distilled water.
12. Add 1 mL of 70% isopropanol and incubate for 5 min.
13. Discard the excess of isopropanol. A PBS wash is not needed at this point.
14. Add 1 mL of Oil-red O solution and incubate for 30 min.
15. Discard the excess of the Oil-red O solution.
16. Rinse with 1 mL of distilled water.
17. Add 500 µL of hematoxylin solution. Incubate for 3 min.
18. Discard the excess of hematoxylin solution.
19. Rinse with 1 mL of distilled water.
20. Observe under the microscope at a magnification of 400x (Objective 40x, Ocular 10x).

Materials

Name	Company	Catalog Number	Comments
Culture media RPMI 1640	ThermoFisher-Gibco, US	31800-022	-
Fetal Bovine Serum (FBS)	ThermoFisher-Gibco, US	A4766801	-
HepG2 cell line	ATCC, US	HB-8065	Hepatocellular carcinoma human cells.
Humidified incubator	Thermo Electronic Corporation,US	Model: 3110	Temperature (37 °C ± 1 °C), humidity (90% ± 5%) , CO2 (5% ± 1%)
Inverted microscope Eclipse	NIKON, JPN	Model: TE2000-S	-
Isopropanol	Sigma-Aldrich, US	I9030-4L	-
Oil Red O Kit	Abcam, US	ab150678	Kit for histological visualization of neutral fat.
Paraformaldehyde	Sigma-Aldrich, US	P6148-500G	-
Penicillin/streptomycin	ThermoFisher-Gibco, US	15140-122	Antibiotics 10,000 U/mL Penicillin, 10,000 μg/mL Streptomycin
Phosphate buffered saline	ThermoFisher-Gibco, US	10010-023	-
Serological Pipettes	Sarstedt, AUS	86.1253.001	Non-pyrogenic, sterile, 5 mL
Serological Pipettes	Sarstedt, AUS	86.1254.001	Non-pyrogenic, sterile, 10 mL
Sodium bicarbonate	Sigma-Aldrich, US	S5761-1KG	Preparation of culture media
Sodium oleate	Santa Cruz Biotechnology, US	sc-215879A	-
Sodium palmitate	Santa Cruz Biotechnology, US	sc-215881	-
Trypsin 0.05% /EDTA 0.53 mM	Corning, US	25-052-Cl	-
24 well cell culture cluster	Corning, US	3524	Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.