To evaluate the lipid accumulation of 3T3-L1 adipocyte, the Oil red O staining was performed. The cells were washed by PBS and fixed in 2.5% glutaraldehyde for 10 min. After washing, the cells were stained by Oil red O solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 15 min. The stained cells were visualized under an optical microscope. Then, the Oil red O stained lipid was extracted by isopropanol and quantified by measuring the absorbance at 492 nm using a microplate reader (Thermo Fisher Scientific, MA, USA).
Oil red o solution
Manufactured by Fujifilm
Sourced in Japan
Oil Red O solution is a staining agent used in histology and cytology for the detection of neutral lipids. It is a fat-soluble dye that selectively stains neutral lipids, making them visible under a microscope. The solution is commonly used in the analysis of tissue samples and cell preparations to identify the presence and distribution of lipid-containing structures.
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Lab products found in correlation
Indomethacin, by Fujifilm (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Isobutyl 1 methylxanthine, by Merck Group (2 mentions)
Alizarin red s solution, by Fujifilm (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
Protein blocker, by Agilent Technologies (1 mentions)
L ascorbic acid phosphate magnesium salt, by Fujifilm (1 mentions)
Cryostat, by Leica (1 mentions)
Tissue tek otc compound, by Sakura Finetek (1 mentions)
Isopropanol, by Fujifilm (1 mentions)
Formaldehyde solution, by Fujifilm (1 mentions)
Phosphate buffered saline (pbs), by Fujifilm (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Texasred conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alizarin red, by Kanto Chemical (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mab1527, by Merck Group (1 mentions)
9 protocols using oil red o solution
1
Quantifying 3T3-L1 Adipocyte Lipid Accumulation
2
Adipogenic and Osteogenic Differentiation of Adipose Stem Cells
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The capacities of passage 3 ASCs to differentiate into adipogenic lineages and osteogenic lineages were evaluated using a previously reported method [23] (link). For adipogenesis, the medium was switched to an adipogenic medium consisting of a complete medium supplemented with 0.5 μmol/L isobutyl-1-methyl xanthine (Sigma–Aldrich, St. Louis, USA), 0.5 μmol/L dexamethasone (Fuji Pharma, Tokyo, Japan), and 50 μmol/L indomethacin (Wako Pure Chemical Industries, Osaka, Japan). After 14 days, the cells were fixed with 4% PFA and stained with fresh Oil Red O solution (Wako Pure Chemical Industries). For osteogenesis, the medium was switched to a calcification medium consisting of a complete medium supplemented with 50 μmol/L ascorbic acid (Wako Pure Chemical Industries), 10 mmol/L β-glycerophosphate (Sigma–Aldrich), and 100 nmol/L dexamethasone. The cells were incubated for 21 days, and then stained with 1% alizarin red S solution. The proliferation capacities of passage 3 ASCs were evaluated according to the previously reported colony-forming unit assay method [23] (link). Briefly, 100 cells were cultured in 60-cm2 dishes for 9 days and stained with crystal violet. Then, proliferation capacity was measured.
3
Multi-Modal Tissue Staining Protocol
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For paraffin sections, tissues were embedded in paraffin after fixation in 4% paraformaldehyde and subjected to hematoxylin and eosin (H&E) and Oil Red O staining. For cryosection analysis, tissues were sectioned, washed with 60% isopropyl alcohol, and then stained for 1 h at 37 °C in Oil Red O solution (Fujifilm, Wako). Oil Red O-positive lipid droplets were quantified using Image J software. For detection of Dscr-1 in the mouse liver, mice were perfused with 2% paraformaldehyde in PBS. Slides containing cryosections were treated with acetone for 10 min, blocked with a protein blocker (DAKO), and stained with our generated anti-Dscr-1 antibody (13 (link)). Zenon labeling technology (Invitrogen) was used to amplify the mouse tissue signal using a mouse monoclonal antibody. To detect 4-HNE in mouse liver, tissue slides were incubated with an anti-4-HNE antibody (JaICA) and subsequently with HRP-conjugated secondary antibody; the slides were then incubated with DAB reagent and hematoxylin counterstain.
4
Osteogenic and Adipogenic Differentiation of MSCs
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To examine osteogenesis, MSCs at passage five were seeded onto 100-mm culture dish at a density of 1000 cells/dish and cultured for 7 day as previously described [15] (link). The medium was replaced with osteoinductive medium, comprising complete medium supplemented with 82 μg/mL l -ascorbic acid phosphate magnesium salt (Wako Pure Chemical Industry, Osaka, Japan), 10 mM β-glycerophosphate (Sigma–Aldrich), and 10 nM dexamethasone (Dexart, Fuji Pharma, Tokyo, Japan), for an additional 14 days. The MSCs were fixed with 4% paraformaldehyde and stained with 1% alizarin red S solution (Wako), and the alizarin red S-positive colonies were counted. To examine adipogenesis, MSCs at passage five were seeded onto 100-mm culture dish at a density of 1000 cells/dish and cultured for seven days, as previously described [15] (link). The medium was replaced with adipoinductive medium, comprising complete medium supplemented with 100 nM dexamethasone, 0.5 mM isobutyl-1-methylxanthine (Sigma–Aldrich), and 50 mM indomethacin (Wako), for an additional 14 days. The MSCs were fixed with 4% paraformaldehyde and stained with fresh Oil Red O solution (Wako), and the Oil Red O-positive colonies were counted.
5
Multilineage Differentiation Assay for MSCs
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To examine osteogenesis, MSCs at passage 5 were seeded onto 100-mm culture dish at a density of 1000 cells/dish and cultured for 7 day as previously described [19] (link). The medium was replaced with osteoinductive medium (CACn417D250, Cell Applications) for an additional 21 days. The MSCs were fixed with 4% paraformaldehyde and stained with 1% alizarin red S solution (Wako pure chemical). To examine adipogenesis, MSCs at passage 5 were seeded onto 100-mm culture dish at a density of 1000 cells/dish and cultured for 7 days, as previously described [19] (link). The medium was replaced with adipoinductive medium (CACn811D250, Cell Applications) for an additional 21 days. The MSCs were fixed with 4% paraformaldehyde and stained with fresh Oil Red O solution (Wako pure chemical).
6
Quantifying Hepatic Lipid Deposition
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To determine lipid deposition, liver tissues obtained from the sacrificed mice from each group were fixed in 10% formalin solution for 12 h at 4°C and treated with 10, 20 and 30% sucrose solution progressively for 4 h each. Then, the tissues were embedded in Tissue-Tek OTC compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and frozen on dry ice. Tissue sections of 5 µm thickness were cut on a cryostat (Leica Microsystems, Tokyo, Japan) and mounted on slides. The sections were stained with 0.18% Oil Red O solution (Wako Pure Chemical Industries, Ltd.) for 5 min at room temperature, rinsed with 60% isopropanol, and then embedded in a water-soluble mounting agent (Mount-Quick-Aqueous; Daido Sangyo Co., Ltd., Saitama, Japan). Tissue sections were photographed and quantitative analysis of stained areas from a total of 12 randomly-selected fields per group were performed using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA). Oil Red O staining for each group was determined as a fold-ratio relative to the stained area measured for the control WT mice administered ND (ND-WT) at each week.
7
Adipogenic Differentiation of Adipose-Derived Stem Cells
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Adipogenic differentiation was induced by culturing the cells for 7 d in adipocyte
differentiation medium (#DM-2; DS Pharma Biomedical Co., Ltd., Osaka, Japan). The cells
were further cultured in adipocyte maintenance medium (#AM-1; DS Pharma Biomedical) for 7
d. Differentiation was confirmed by Oil Red O staining of intracellular lipid droplets.
Differentiated Adipose-derived Stem Cells (ASCs) were fixed in a 10% formaldehyde solution
(Wako, Osaka, Japan) in phosphate-buffered saline (PBS; Wako) for at least 10 min, washed
with 60% isopropanol (Wako), and stained with Oil Red O solution (Wako) for 10 min
followed by repeated washing with water and destaining in 100% isopropanol for 1 min25 (link),26 (link).
differentiation medium (#DM-2; DS Pharma Biomedical Co., Ltd., Osaka, Japan). The cells
were further cultured in adipocyte maintenance medium (#AM-1; DS Pharma Biomedical) for 7
d. Differentiation was confirmed by Oil Red O staining of intracellular lipid droplets.
Differentiated Adipose-derived Stem Cells (ASCs) were fixed in a 10% formaldehyde solution
(Wako, Osaka, Japan) in phosphate-buffered saline (PBS; Wako) for at least 10 min, washed
with 60% isopropanol (Wako), and stained with Oil Red O solution (Wako) for 10 min
followed by repeated washing with water and destaining in 100% isopropanol for 1 min25 (link),26 (link).
8
Immunocytochemistry and Histochemical Staining
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Colony fixation, permeabilization, and blocking were performed as described (Motohashi et al., 2014 (link)). Primary antibodies, diluted in 0.5% BSA PBS, were then added and allowed to react at room temperature. After having been washed with PBS, the cells were stained with the secondary antibodies in the same manner. Primary antibodies: anti-mouse neuronal class III β-tubulin (1:500; TuJ-1, Covance), anti-mouse glial fibrillary acidic protein (GFAP, 1:500; Z0334, DakoCytomation), anti-mouse α smooth muscle actin (1:500; 1A4, Sigma), anti-mouse peripherin (1:100; MAB1527, Chemicon), anti-nestin (1:500; Rat401, Chemicon), and anti-S100β (1:100, Sigma, SH-B1). Secondary antibodies: Texas Red-conjugated anti-mouse IgG (1:500; Molecular Probes) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:500; Molecular Probes). Nuclei were stained with Hoechst 33258 (Sigma). Colonies were examined by using an Olympus IX-71 fluorescence microscope.
ALP staining was performed with an ALP staining kit (Muto Chemical Co.). Regarding Alizarin Red staining, cells were fixed in methanol at 4°C for 20 min, and Alizarin Red (Kanto Chemical) staining was then carried out for 5 min at room temperature. For Oil Red O staining, cells were fixed in 4% paraformaldehyde for 15 min and then stained with 60% Oil Red O solution (Wako) for 30 min at room temperature.
ALP staining was performed with an ALP staining kit (Muto Chemical Co.). Regarding Alizarin Red staining, cells were fixed in methanol at 4°C for 20 min, and Alizarin Red (Kanto Chemical) staining was then carried out for 5 min at room temperature. For Oil Red O staining, cells were fixed in 4% paraformaldehyde for 15 min and then stained with 60% Oil Red O solution (Wako) for 30 min at room temperature.
9
Artificial Oily Dirt Removal Evaluation
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Artificial oily dirt was prepared according to a slightly modified version of a procedure often used in detergency tests of commercial household detergents [33, 34] . In this study, artificial oily dirt was used for testing the detergency of the original commercial denture cleaners or was modified by adding detergents to the commercial denture cleaners. The dirt was prepared by mixing 80 g beef tallow oil (Yamakei Industrial Co., Ltd, Osaka, Japan), 40 g soybean oil (Yamakei Industrial Co., Ltd, Osaka, Japan), 1 g monoolein (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), 0.4 g Oil Red O solution (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), and 120 mL chloroform (purity +99%; Fujifilm Wako Pure Chemical Corp., Osaka, Japan). The experimental method is shown in Figure 1C. Briefly, a fixed volume (3 μL) of the prepared oily dirt was spread on transparent acrylic resin plates and dried at 23 ± 2 °C overnight. Subsequently, the plates were immersed into the control and denture cleaner solutions, with and without surfactants at 0.5%, and were maintained at 23 ± 2 °C for up to 24 h. The plates were examined at 1, 3, 6, 12, and 24 h after the immersion. The time required for the complete removal of artificial dirt spots was determined by calculating the mean and SD of the times for six specimens, for each solution (Table 3).
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