Paraffin-embedded sections were stained with hematoxylin and eosin (200108, Muto Pure Chemicals, Tokyo, Japan) or sirius red (MKCB3138V, Sigma-Aldrich, Tokyo, Japan). For lipid staining, frozen sections were stained with Oil Red O (M3G0644, NACALAI TESQUE, Kyoto, Japan). Macrophages were detected by F4/80 (MCA497R, Bio-Rad, Tokyo, Japan) and VECTASTAIN secondary antibodies (Vector laboratories, Burlingame, USA). To quantify the area of staining by Oil Red O and Sirius Red, images of five random fields from each section were processed with Image J software (National Institute of Mental Health, Bethesda, MD, USA). Each value was expressed as the percentage of the total area of the section. Numbers of F4/80 positive cells were counted and averaged for five random fields of each section.
Oil red o
Manufactured by Nacalai Tesque
Sourced in Japan
Oil Red O is a lipophilic (fat-soluble) dye used in histological and cytological procedures to stain neutral triglycerides and lipids. It is commonly used to detect the presence of lipid droplets in tissues and cells.
Automatically generated - may contain errors
Lab products found in correlation
F4 80 mca497r, by Bio-Rad (2 mentions)
Vectastain secondary antibodies, by Vector Laboratories (2 mentions)
Mkcb3138v, by Merck Group (2 mentions)
Hematoxylin and eosin, by Muto Pure Chemicals (1 mentions)
Osteogenic differentiation medium, by PromoCell (1 mentions)
Adipocyte differentiation medium, by Cell Applications (1 mentions)
Bcip nbt solution kit for alkaline phosphatase stain, by Nacalai Tesque (1 mentions)
Haematoxylin and eosin, by Muto Pure Chemicals (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Dm5000b microscope, by Leica (1 mentions)
5 protocols using oil red o
1
Histological Assessment of Tissue Composition
2
Quantitative Lipid Droplet Assay
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As described above, sorted cells were treated with differentiation medium. Two days later, the medium was replaced with DMEM-10% FBS. The cells were cultured for 2 more days before they were fixed with 10% formaldehyde (Nacalai Tesque) for 10 min. The fixed cells were washed twice with PBS and with 60% isopropanol for 1 min, and incubated in Oil Red O (Nacalai Tesque) solution in 60% isopropanol for 15 min. Following the removal of the Oil Red O solution, the stained cells were washed once with 60% isopropanol and twice with PBS. PBS was added to each well, and images of stained cells were acquired using a BioZero microscope BZ-9000 (Keyence, Osaka, Japan). To quantify the Oil Red O staining, the PBS was discarded, and the wells were completely dried and incubated with 100% isopropanol for 15 min with gentle agitation. The OD at 490 nm was measured.
3
Osteoblast and Adipocyte Differentiation
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ASCs were differentiated into osteoblasts in osteogenic differentiation medium (PromoCell, Heidelberg, Germany). Alkaline phosphatase of differentiated cells was visualized using the BCIP-NBT Solution Kit for Alkaline Phosphatase Stain (Nacalai), and differentiated into mature adipocytes in adipocyte differentiation medium (Cell Applications Inc.). Lipid accumulation in adipocytes was visualized by Oil Red O (Nacalai) according to the manufacturer’s protocol.
4
Histological Staining and Quantification
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Paraffin-embedded sections were stained with haematoxylin and eosin (200,108, Muto Pure Chemicals, Tokyo, Japan) or Sirius Red (MKCB3138 V, Sigma–Aldrich, Tokyo, Japan). For lipid staining, frozen sections were stained with Oil Red O (M3G0644, Nacalai Tesque, Kyoto, Japan). Macrophages were detected by F4/80 (MCA497R, Bio–Rad, Tokyo, Japan) and Vectastain secondary antibodies (Vector Laboratories, Burlingame, CA, USA). To quantify the area of staining by Oil Red O and Sirius Red, images of 5 random fields from each section were processed using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA). Each value was expressed as the percentage of the total area of each section that stained positively. The number of F4/80-positive cells was counted and averaged for 5 random fields in each section. The NAS and fibrosis stages were scored by Y.K. in a blinded manner according to the method of Kleiner et al.44 (link).
5
Lipid Droplet Visualization in HeLa Cells
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HeLa cells were grown on coverslips and transfected with the indicated plasmids. Cells were fixed with 3.7% formaldehyde in PBS for 10 min at 37 °C and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. After washing with 60% isopropanol, cells were incubated with 1.8 mg/ml oil red O (Nacalai Tesque, Kyoto, Japan) in 60% isopropanol for 20 min at room temperature, followed by repeat washing with PBS. Cells were then blocked with blocking solution (10 mg/ml BSA in PBS) at 37 °C for 30 min and incubated with anti-FLAG M2 antibody (2 μg/ml) in blocking solution for 1 h at room temperature. After washing three times with PBS, the cells were incubated with Alexa Fluor 488-or 594-conjugated anti-mouse antibody (each at 5 μg/ml; Molecular Probes, Life Technologies, Eugene, OR) in blocking solution for 1 h at room temperature. Coverslips were washed with PBS, mounted with Prolong Gold Antifade Reagent (Molecular Probes, Life Technologies) and observed under a Leica DM5000B microscope (Leica Microsystems, Wetzlar, Germany). In the case of EGFP-fusion protein-expressing cells, the permeabilization, blocking, and antibody staining steps were omitted.
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