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

Protocolo

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).

Materiales

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.