Cells were plated at 1 × 105 cells/cm2 in 6-well plates and then induced in an adipogenic medium for 14 days, which was renewed every two days. The induction medium was: a complete medium supplemented with 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich), 10 µM insulin (Actrapid®; NovoNordisk A/S, Bagsværd, Denmark) and 200 µM indomethacin (Sigma-Aldrich). Cells were subsequently cultured for five more days in a complete medium supplemented with 10 µM insulin (maintenance medium). After fixing the cells with 4% paraformaldehyde for 30 min at room temperature, lipid accumulation was visualized through Oil Red O (ORO) staining (Sigma-Aldrich) at 0.3% in 60% isopropanol for 30 min using gentle agitation (Zuk et al., 2001 (link)).
Oil red o staining
Manufactured by Merck Group
Sourced in United States, Germany, China
Oil Red O is a fat-soluble dye used for staining neutral lipids and triglycerides in histological and cytological preparations. It is commonly used to visualize lipid accumulation in cells and tissues.
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Lab products found in correlation
Indomethacin, by Merck Group (19 mentions)
Dexamethasone (dex), by Merck Group (17 mentions)
Insulin, by Merck Group (15 mentions)
Alizarin red s staining, by Merck Group (13 mentions)
Alizarin red staining, by Merck Group (11 mentions)
Alizarin red, by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Alcian blue staining, by Merck Group (8 mentions)
Isobutylmethylxanthine, by Merck Group (7 mentions)
Hydrocortisone, by Merck Group (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
β glycerophosphate, by Merck Group (4 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Isopropanol, by Merck Group (3 mentions)
Alizarin red s, by Merck Group (3 mentions)
High glucose dmem, by Thermo Fisher Scientific (3 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Hematoxylin, by Merck Group (2 mentions)
140 protocols using oil red o staining
1
Adipogenic Differentiation of Cells
2
Visualizing Lipid Droplets in Frozen Tissues
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Frozen liver and BAT of rats were sectioned 10 μm thick using a cryostat section and fixed with 10% formalin for 30 min, respectively. Lipid droplets were detected by Oil-Red-O (ORO) staining (Sigma-Aldrich, USA). Sections were stained for 15 min in ORO solution and counterstained with hematoxylin (Sigma-Aldrich, USA) for 30 s. The images were photographed by inverted microscope (Olympus, Japan).
3
Quantifying Aortic Lymphatic Capillaries
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The heart and aorta were removed and fixed in 4% PFA for 2 hours.The heart was transferred into PBS containing 30% sucrose (wt/vol) overnight at 4 °C before being immersed in OCT compound and stored at −80 °C. Eight-micrometer-thick cryosections of the aortic sinus were prepared. Cross-sections of the aortic sinus were stained with anti-LYVE-1 (ABCAM) and anti-CD68 (Biolegend) antibodies, and then incubated with the appropriate secondary antibodies. As macrophages can also be positive for LYVE-1, adventitial lymphatic capillaries were identified as LYVE-1+ CD68− cells forming vessel-like shapes. Whole-mount immunohistochemical analysis of the ear dermis to visualize lymphatic vessels was performed as described previously47 (link). Ear dermis were stained for lymphatic capillaries (anti-LYVE-1, ABCAM) at 4 °C, and then sections were incubated with Alexa Fluor 647 conjugated donkey anti-rabbit antibody and Cy3 donkey anti-rat (Jackson ImmunoResearch). All imaging was performed on a Fluoview FV10i (Olympus). All vessel counts were performed by one observer. The relative quantification of the number of lymphatic capillaries (LYVE-1+ vessels), their diameter and the total surface area they occupy was determined by computer-assisted morphometric analysis. Neutral lipid assessment in atherosclerotic lesions was performed by Oil-red-O (ORO) staining (Sigma).
4
Quantitative Adipogenic Differentiation Assay
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Adipogenic differentiation was evaluated via Oil-Red-O (ORO) staining (Sigma-Aldrich, MA, USA). Cells were washed twice with PBS and then fixed with 10% formalin (Sigma-Aldrich, MA, USA) for 10 min. After fixation, ORO staining solution was applied at room temperature (RT) for 30 min to stain the lipid vesicles. To perform the quantification of the remaining ORO, the dye was eluted using isopropanol (Sigma-Aldrich, MA, USA) containing 4% igepal (Sigma-Aldrich, MA, USA), and the absorbance was measured at 492 nm14 (link).
5
Quantifying Intracellular Lipid Accumulation
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Cardiac lipid accumulation is associated with decreased cardiac function. To quantify lipid accumulation, Oil red O (ORO) staining (Sigma-Aldrich, St Louis, MO, USA), a widely used lysochrome (fat-soluble) diazo dye for staining and quantifying neutral intracellular lipids, was adapted as previously described [22 (link)]. Briefly, following the relevant treatment conditions, H9c2 cells were fixed with 4% (v/v) paraformaldehyde for 15 min and washed three times in PBS. Thereafter, cells were incubated in freshly prepared 0.7% (v/v) ORO staining solution for 30 min at room temperature. Following a 5 min rinse in distilled water, cells were immersed in 100% isopropanol for 20–30 s to extract the dye. Supernatants were then transferred to a 96-well plate for measurement of optical density for lipid accumulation at 490 nm with a BioTek EL×800 plate reader using Gen 5 software. For normalisation, cells were treated with 70% ethanol, then the solution was aspirated before the addition of 400 µL crystal violet (CV) for 5 min at room temperature. The CV was removed, and cells washed with PBS. Thereafter, 70% ethanol was added to extract the CV and 100 µL transferred to a clean 96-well plate for the measurement of optical density at 570 nm.
6
Liver Mass Index and Lipid Analysis
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At the end of the study, the mice were sacrificed to measure liver mass and liver mass index by using the following formula: live mass index = liver mass/body mass × 100%. Afterward, the liver samples were immersed in 10% formalin neutral buffer solution for 48 h, then processed routinely, embedded in paraffin, sectioned to 5 μm thickness and stained with hematoxylin and eosin (H&E). Lipid accumulation in liver was analyzed by Oil red O (ORO) staining (Sigma). Slides were observed with a light microscope (Leica DMi 8).
7
Visualization of Neutral Lipids in Zebrafish Embryos
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Neutral lipids were visualized by Oil Red O (ORO) staining (Sigma-Aldrich), according to the protocol of Yoganantharjah et al. [69 (link)]. At 144 hpf, the control and treated embryos were anesthetized with 0.4% Tricaine, washed and soaked in fish water, fixed in 4% paraformaldehyde for 12 h at 4°C, and washed three times in phosphate-buffered saline (PBS) 1X solution. The fish were then preincubated in 60% isopropanol in PBS 1× solution for 30 min, dyed with fresh 0.3% ORO (Sigma Aldrich) for 3 h, and thoroughly washed with 60% isopropanol in PBS 1X solution to minimize background. The stained embryos were then observed, and images were obtained with a Zeiss Axiozoom V13 (Carl Zeiss AG) microscope, equipped with a PlanNeoFluor Z 1×/0.25 FWD 56 mm lens at 20× magnification.
8
Lipid Accumulation Quantification in hBM-MSCs
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The lipid accumulation was primarily assessed by Oil Red O (ORO) staining (Sigma-Aldrich). Differentiated adipocytes were rinsed twice with phosphate-buffered saline (PBS) and fixed with 10% formalin in PBS (pH 7.4) for 1 h. Fixed cells were washed once with 60% isopropanol and stained with 0.2% ORO solution for 10 min at 24°C and washed four times with tap water. To visualize the nucleus, differentiated hBM-MSCs were counterstained with hematoxylin reagent (Sigma-Aldrich) for 1 min and then washed four times with tapping water. The ORO-stained hBM-MSCs were observed using an inverted phase-microscope (Nikon Co., Tokyo, Japan).
9
Lipid Accumulation Quantification in Adipocytes
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Lipid accumulation in 3T3-L1 adipocytes was measured using Oil Red O staining (# O1391, Sigma-Aldrich, Canada) as per the protocol. Oil Red O-stained cells were viewed using a Leica DMIL-LED Microscope at 400× magnification, and Infinity Camera Analyze Software (version 6.5.5) was used to capture the images. Oil Red O-dye was extracted from cells by adding isopropanol, the absorbance of extracted dye was measured using a spectrophotometer at 520 nm, and isopropanol was used as a blank.
10
Multilineage Differentiation of Red Panda eMSCs
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For adipocytic differentiation, eMSCs at passage 4 were seeded in 6-well plates and treated with adipogenic medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 8 days, adipogenesis was evaluated by Oil red O staining (Sigma). Staining was assessed by bright-field inverted microscopy (IX73, Olympus). Red panda eMSCs cultured in normal growth medium served as control.
For chondrogenic differentiation, eMSCs at passage 4 at a density of 4 × 105 cells were cultured in chondrogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 21 days, chondrogenesis was detected by the staining of toluidine blue. Red panda eMSCs cultured in normal growth medium served as control.
For hepatogenic differentiation, eMSCs were cultured in hepatogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 16 days, hepatogenic differentiation was evaluated by cytokeratin 18 (CK18) (ab181597, Abcam) immunofluorescence staining, detection of CK 18, ALB and DKK1 mRNA expression, ALB protein expression, Periodic Acid-Schiff (PAS) staining and Indocyanine Green (ICG) uptake. Red panda eMSCs cultured in normal growth medium served as control.
For chondrogenic differentiation, eMSCs at passage 4 at a density of 4 × 105 cells were cultured in chondrogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 21 days, chondrogenesis was detected by the staining of toluidine blue. Red panda eMSCs cultured in normal growth medium served as control.
For hepatogenic differentiation, eMSCs were cultured in hepatogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 16 days, hepatogenic differentiation was evaluated by cytokeratin 18 (CK18) (ab181597, Abcam) immunofluorescence staining, detection of CK 18, ALB and DKK1 mRNA expression, ALB protein expression, Periodic Acid-Schiff (PAS) staining and Indocyanine Green (ICG) uptake. Red panda eMSCs cultured in normal growth medium served as control.
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