To visualize lipid droplets, the HL-1 atrial myocytes were stained with Oil Red O (ScyTek Laboratories, Logan, UT, USA) according to the manufacturer’s directions. Sections were mounted and visualized with a Dmi3000 microscope. The Oil Red O-stained area per myocyte was analyzed by Cellsens Dimension (Olympus, Tokyo, Japan), with at least 60 randomly-chosen myocytes for each experiment. Stretched and non-stretched myocytes were compared in five experiments.
Oil red o
Manufactured by ScyTek Laboratories
Sourced in United States
Oil Red O is a fat-soluble dye used for staining neutral lipids and lipoproteins in fixed frozen sections and cell preparations. It has a strong affinity for neutral fats, making it a useful tool for the detection and visualization of lipid-containing structures.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o, by Olympus (2 mentions)
Cellsens dimension, by Olympus (2 mentions)
Alizarin red, by IHC World (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Hematoxylin, by ScyTek Laboratories (1 mentions)
Mayer s hematoxylin, by ScyTek Laboratories (1 mentions)
Bluing reagent, by ScyTek Laboratories (1 mentions)
Oil red o, by Merck Group (1 mentions)
Stempro adipogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Hm340e, by Thermo Fisher Scientific (1 mentions)
Direct red 80, by Merck Group (1 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Bx53f, by Olympus (1 mentions)
Cellsens standard, by Olympus (1 mentions)
6 protocols using oil red o
1
Visualizing Lipid Droplets in Atrial Myocytes
2
Multilineage Differentiation of hFDFs and BM-MSCs
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hFDFs were cultured in parallel with BM-MSCs with three technical repeats for each
lineage to determine osteogenic, adipogenic, and chondrogenic differentiation (LONZA). For
osteogenic differentiation, cells received the induction of B-glycerophosphate and
dexamethasone, and calcium deposition was detected by Alizarin Red (IHC World, Woodstock,
MD, USA). Adipogenic differentiation was triggered with dexamethasone in combination with
IBMX (3-isobutyl-1-methylxanthine), and oil droplets were stained with Oil Red O (Scytek,
Logan, UT, USA). Chondrogenic differentiation was induced with TGF-β3, and the cells were
centrifuged and cultured as a pellet. After 2–3 weeks, pellets were stained with Alcian
blue for proteoglycans (Scytek).
lineage to determine osteogenic, adipogenic, and chondrogenic differentiation (LONZA). For
osteogenic differentiation, cells received the induction of B-glycerophosphate and
dexamethasone, and calcium deposition was detected by Alizarin Red (IHC World, Woodstock,
MD, USA). Adipogenic differentiation was triggered with dexamethasone in combination with
IBMX (3-isobutyl-1-methylxanthine), and oil droplets were stained with Oil Red O (Scytek,
Logan, UT, USA). Chondrogenic differentiation was induced with TGF-β3, and the cells were
centrifuged and cultured as a pellet. After 2–3 weeks, pellets were stained with Alcian
blue for proteoglycans (Scytek).
3
Quantifying Myocardial Lipid Accumulation
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Left atrial tissues were sliced into 8-µm sections, stained with Oil red O to visualize lipid accumulation (ScyTek Laboratories, Utah, USA) according to the manufacturer’s directions. Sections were mounted and visualized using an Olympus BX51 microscope. The Oil red O stained area per myocyte was analyzed by Cellsens Dimension (Olympus, JAPAN) with at least 100 randomly chosen myocytes per each sample.
4
Fatty Acid Profiling and Oil Red-O Staining
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Direct fatty acid methyl ester (FAME) synthesis and the gas chromatography/mass spectrometry method previously described [27 (link),28 (link)] were utilized to identify liver fatty acid concentrations and to validate EPA delivery to the liver. Frozen sections from the harvested livers were routinely fixed and stained with Oil Red-O (Sigma-Aldrich, St. Louis, MO, USA) and counterstained with Mayer’s hematoxylin (ScyTek Laboratories Inc., Logan, Utah, USA). To perform the staining, the slides were immersed in formalin for 10 min, dipped in 60% isopropanol, covered in Oil Red-O for 15 min, dipped in 60% isopropanol, rinsed in water, counterstained with hematoxylin for 3 min, immersed briefly in Bluing Reagent (ScyTek Laboratories Inc., Logan, UT, USA), rinsed with water, and mounted. Lipids were imaged at 20× magnification using a cell-imaging microscope. Liver TAGs were measured as described previously (L-Type TAG M kit, Wako Chemicals USA, Inc., North Chesterfield, VA, USA) and normalized to liver weight [16 (link),29 (link)].
5
Adipogenic Differentiation of fWJ-MSCs
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To determine if the fWJ-MSCs could differentiate into adipocytes, the cells were treated with adipogenic differentiation media (StemPro Adipogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA, USA). At passage 5, when cells reached confluency of 80 to 90%, the medium was changed to the adipogenic differentiation medium, and the cells were incubated for 3 weeks, changing media once every 3 days. For comparison, passage 5 cells were used as undifferentiated cells. After 3 weeks, Oil Red O staining (Scytek, Logan, UT, USA) was performed to detect lipid droplets. Cells were fixed with 10% neutral buffered formalin for fixation at least 1 h and rinsed with 60% isopropanol prior to incubation in freshly diluted Oil Red O for 10 min. Stains were solubilized using 100% isopropanol, and the resulting absorbance was measured at 500 nm using a spectrophotometer.
6
Histological Evaluation of NASH Progression
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Liver samples of each experimental group were carefully removed and immersed in 10% formalin for tissue fixation. Paraffin-embedded liver was sectioned by a microtome (HM-340E, Thermo Fisher Scientific, MA, USA). We stained the sections with hematoxylin & eosin (H&E) in accordance with typical protocol. To confirm NASH severity, an NAFLD activity score (NAS) system was obtained in accordance with the criteria by Kleiner et al [22 (link)].
To detect the apoptosis in the liver, we carried out a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay on the paraffin-embedded sections, using an ApopTag Peroxidase in situ apoptosis detection kit (EMD Millipore, CA, USA).
To assess the severity of fibrosis, we did sirius red staining of the liver sections with Direct Red 80 (Sigma-Aldrich). We also did Oil-Red O staining (ScyTek Laboratories, UT, USA) using frozen liver tissue sections or cultured cells to determine intracellular lipid accumulation. We evaluated the stained liver section or cell images using light microscopy (BX53F, Olympus Corp., Tokyo, Japan) and digital image software (cellSens Standard, Olympus Corp.). The data is expressed as the mean of each positively stained area per field in the livers or cells.
To detect the apoptosis in the liver, we carried out a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay on the paraffin-embedded sections, using an ApopTag Peroxidase in situ apoptosis detection kit (EMD Millipore, CA, USA).
To assess the severity of fibrosis, we did sirius red staining of the liver sections with Direct Red 80 (Sigma-Aldrich). We also did Oil-Red O staining (ScyTek Laboratories, UT, USA) using frozen liver tissue sections or cultured cells to determine intracellular lipid accumulation. We evaluated the stained liver section or cell images using light microscopy (BX53F, Olympus Corp., Tokyo, Japan) and digital image software (cellSens Standard, Olympus Corp.). The data is expressed as the mean of each positively stained area per field in the livers or cells.
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