To confirm differentiation, cells were stained with Oil Red O. Following treatments as indicated, the cells were washed twice with phosphate-buffered saline (PBS) and fixed in 3.7% formaldehyde for 1 h. After aspiration of the formaldehyde, the cells were stained with Oil Red O for 1 h. Oil Red O was prepared by dissolving 0.5 g Oil Red O (Sigma-Aldrich) in 100 ml 2-propanol and diluting it with water (6:4), followed by filtration. Stained cells were washed carefully in PBS and covered with water until photographed using the 20X lens of a Leica DMI 6000 microscope.
Oil red o
Manufactured by Merck Group
Sourced in United States, Germany, China, Japan, United Kingdom, Sao Tome and Principe, Italy, Macao, Australia, France, Switzerland, Spain, India, Poland, Canada
Oil Red O is a fat-soluble dye used in histology and cell biology for the staining of neutral lipids, such as triglycerides and cholesterol esters. It is a useful tool for the identification and visualization of lipid-rich structures in cells and tissues.
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Lab products found in correlation
Dexamethasone (dex), by Merck Group (285 mentions)
Insulin, by Merck Group (228 mentions)
Indomethacin, by Merck Group (181 mentions)
Alizarin red s, by Merck Group (160 mentions)
Alizarin red, by Merck Group (129 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (102 mentions)
β glycerophosphate, by Merck Group (82 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (81 mentions)
Alcian blue, by Merck Group (78 mentions)
Isobutylmethylxanthine, by Merck Group (57 mentions)
Ascorbic acid, by Merck Group (50 mentions)
Isopropanol, by Merck Group (47 mentions)
Hematoxylin, by Merck Group (43 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (41 mentions)
Paraformaldehyde (pfa), by Merck Group (40 mentions)
Rosiglitazone, by Merck Group (31 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (29 mentions)
β glycerol phosphate, by Merck Group (29 mentions)
Stempro adipogenesis differentiation kit, by Thermo Fisher Scientific (28 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (26 mentions)
2 545 protocols using oil red o
1
Oil Red O Staining for Adipogenesis
2
Quantifying Intracellular Lipid Accumulation
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Intracellular lipid accumulation was measured using Oil Red O. The Oil Red O working solution was prepared as described by Ramírez-Zacarías et al. [26 (link)]. Briefly, Oil Red O stock solution was prepared Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) dissolved in isopropanol at the concentration of 3.5 mg/mL, and the Oil Red O working solution was prepared 60% Oil Red O stock solution mixed with 40% distilled water. 3T3-L1 adipocytes were harvested 6 days after the initiation of differentiation. Cells were washed twice with phosphate buffered saline (PBS, pH 7.4) and then fixed with 10% neutral formalin for 2 hours at room temperature. After washing with 60% isopropanol, the cells were stained with Oil Red O working solution for 30 min and then were washed 4 times with water in order to remove the unbound dye. The stained cells were observed by an Olympus IX71 Research Inverted Phase microscope (Olympus Co., Tokyo, Japan). Following the microscopic observation, 100% isopropanol was added as an extraction solution to extract the staining dye of cells. The absorbance of the extracted dye was measured spectrophotometrically at 500 nm in a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).
3
Quantitative Lipid Droplet Analysis via Oil Red O Staining
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Cytoplasmic lipid droplets were stained with oil red O, as described in a previous report [12 (link)]. Briefly, cells were rinsed three times in phosphate-buffered saline (PBS) and then fixed in 10% (v/v) formaldehyde for 10 min. The cells were washed twice with PBS, followed by staining for 30 min at 37℃ in freshly diluted oil red O (Sigma Chemical Co., St. Louis, MO, USA) solution (six parts oil red O stock and four parts H2O; oil red O stock solution is 0.5% oil red O in isopropanol), followed by further washing with PBS. The stained cytoplasmic triglycerides were visualized and photographed using a microscope. For quantification of oil red O content, the cells were washed 3 times with distilled H2O for removal of background staining, and isopropanol was added to resolve oil red O. The OD510 nm of the de-staining isopropanol was measured by spectrophotometry.
4
Histological Analysis of Liver Lipid Depots
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Liver samples were analysed for lipid and fat depots by Oil red O staining. Frozen samples were cut into 30 µm-thick sections using a sliding microtome (Leica SM200R, Wetzlar, Germany) and fixed with 10% formal calcium. Sections were washed with distilled water and rinsed with 60% isopropanol. Then, sections were stained with freshly prepared Oil red O (Sigma, St Louis, MO) working solution for 20 minutes (Oil red O stock stain: 0.5% of Oil red O in isopropanol; Oil red working solution: 30 ml of the stock stain and 20 ml of distilled water). Sections were rinsed with 60% isopropanol, counterstained with Mayer’s hematoxylin, rinsed with tap water and mounted in aqueous media.
5
Liver Lipid Extraction and Visualization
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Fat extraction was performed via organic solvent-based extraction. Briefly, total lipids from frozen liver samples were extracted with chloroform/methanol (2:1, v/v) and butylated hydroxytoluene (0.025%, w/v). The lower phase containing lipids was separated after 2 centrifugation steps (2800× g, 4 °C for 10 min) and dried by nitrogen. The liver fat content was expressed as a percentage of the tissue weight.
The liver samples were analyzed for fat depots via oil red O staining. The frozen samples were cut into 30 µm-thick sections using a sliding microtome (Leica SM200R, Wetzlar, Germany) and fixed with 10% formal calcium. The sections were washed with distilled water and rinsed with 60% isopropanol. Then, the sections were stained with freshly prepared oil red O (Sigma, St Louis, MO, USA) working solution for 20 min (oil red O stock stain: 0.5% of oil red O in isopropanol; Oil red working solution: 30 mL of the stock stain and 20 mL of distilled water). The sections were rinsed with 60% isopropanol, counterstained with Mayer’s hematoxylin, rinsed with tap water, and mounted in aqueous media.
The liver samples were analyzed for fat depots via oil red O staining. The frozen samples were cut into 30 µm-thick sections using a sliding microtome (Leica SM200R, Wetzlar, Germany) and fixed with 10% formal calcium. The sections were washed with distilled water and rinsed with 60% isopropanol. Then, the sections were stained with freshly prepared oil red O (Sigma, St Louis, MO, USA) working solution for 20 min (oil red O stock stain: 0.5% of oil red O in isopropanol; Oil red working solution: 30 mL of the stock stain and 20 mL of distilled water). The sections were rinsed with 60% isopropanol, counterstained with Mayer’s hematoxylin, rinsed with tap water, and mounted in aqueous media.
6
Adipogenic Differentiation Assay Protocol
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To induce adipogenesis, cells were cultured for 3 weeks in adipogenic media (DMEM (Gibco, 41-965-062), 100 U/ml penicillin +100 mg/ml streptomycin (Gibco, 15140-122), 10% fetal bovine serum, 0.2 mM Indomethacin (Sigma, 57413), 0.5 mM IBMX (Sigma, I5879), 10−6 M dexamethasone (Sigma, D8893), 10 ug/ml insulin (human, Sigma, I9278). Cells were seeded at a density of 15,000 cells/cm2; media was refreshed twice per week. To visualize lipid formation, cells were stained with Oil Red O as described before (De Boer et al., 2004 (link)). Briefly, cells were fixed with 10% formalin for 30 min at room temperature, rinsed with a water and washed with 60 % isopropanol. The sample was stained for 5 min in freshly filtered Oil Red O solution (stock: 500 mg Oil Red O (Sigma, O0625), 99 ml isopropanol, 1 ml water; stain: 15 ml stock + 10 ml water).
Oil Red O staining was quantified by extraction with 1 ml of 4% Igepal (Sigma, 56741) in isopropanol for 15 min by shaking at room temperature. Absorbance was measured at 540 nm.
Oil Red O staining was quantified by extraction with 1 ml of 4% Igepal (Sigma, 56741) in isopropanol for 15 min by shaking at room temperature. Absorbance was measured at 540 nm.
7
Oil Red O Staining for Lipid Droplets
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Dishes were washed twice with PBS and cells were fixed in 3.7% formaldehyde for 1 h. After aspiration of the formaldehyde, the cells were stained with Oil red O for 1 h. Oil red O was prepared by dissolving 0.5 g Oil red O (Sigma-Aldrich) in 100 ml 2-propanol (Sigma-Aldrich) and diluting it 3:2 with water, followed by filtration. After staining, dishes were washed carefully with PBS and covered with water until photographed.
8
Oil Red O Staining of Skin Tissues
9
Lipid Accumulation Quantification by Oil Red O
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Lipid accumulation was detected at day 21 by oil red O (Sigma-Aldrich). Cells were fixed in 10% formalin for 2 h at room temperature, washed with isopropanol 60% (Merck-Germany) and stained with 0.3% oil red O (Sigma-Aldrich) for 10 min. For qualitative analysis, culture images were captured with a high-resolution digital camera (Canon EOS Digital Rebel Camera, Canon) and the lipid accumulation was measured using a colorimetric assay. The incorporated oil red O (Sigma-Aldrich) was extracted by incubation with 100% isopropanol for 10 min under shaking at room temperature. After appropriate dilution, this solution was read at 500 nm in a plate reader μQuant (BioTek), and the data were obtained in quintuplicate (n=5) and expressed as absorbance.
10
Lipid Droplet Staining with Oil Red O
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Cells were washed in 1X PBS and fixed in 3.7% formaldehyde solution for 1 h, followed by staining with Oil Red O for 1 h. Oil Red O was prepared by diluting a stock solution (0.5 g of Oil Red O (Sigma) in 100 ml of isopropanol) with water (6:4) followed by filtration. After staining, plates were washed twice with water and photographed using an iPhone.
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