Fetal and newborn mouse lungs were dissected, fixed in 4% paraformaldehyde (Electron Microscopy Sciences) and paraffin embedded. Sections were then stained with hematoxylin (Dako North America), eosin (Ricca Chemical), modified Hart's stain (reagents from Electron Microscopy Sciences) or Oil Red O (IHC World). Volumetric measurement of lung tissue and airspace volume of hematoxylin and eosin-stained section was performed using ImageJ. For immunostaining, fetal mouse lungs were fixed in 4% paraformaldehyde, washed and processed through a sucrose gradient before being embedded in OCT freezing media (Tissue-Tek; Sakura Finetek USA). Frozen sections were stained with antibodies of interest followed by Alexa-conjugated secondary antibodies, and nuclei were stained with DRAQ5.
Oil red o
Manufactured by IHC World
Sourced in United States
Oil Red O is a fat-soluble dye used in histology and cell biology to stain neutral lipids and lipoproteins. It is commonly used to identify the presence of lipids in tissue sections or cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Advancestem adipogenic differentiation medium, by Thermo Fisher Scientific (2 mentions)
Advancestem stem cell growth supplement, by Thermo Fisher Scientific (2 mentions)
Hematoxylin, by Agilent Technologies (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Hematoxylin, by Sakura Finetek (1 mentions)
Alcian blue, by IHC World (1 mentions)
Alizarin red, by IHC World (1 mentions)
Tgf β3, by Lonza (1 mentions)
4 protocols using oil red o
1
Histological Analysis of Fetal and Newborn Mouse Lungs
2
Adipogenic Differentiation of hUC-MSCs
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hUC-MSCs at passage 3 were seeded in six-well plates in complete medium in triplicate at a final density of 5000 cells per cm2 according to the previous report with some modifications [30 (link)] Forty-eight hours later, designated as day 0, differentiation was initiated using the adipogenic induction medium (AdvanceSTEM adipogenic differentiation medium supplemented with 10% AdvanceSTEM stem cell growth supplement, Thermo Scientific, Rockford, IL), according to the manufacturer's instructions. The medium was changed every 3-4 days, and the experiment was terminated after 3 weeks. The differentiated hUC-MSCs were fixed with 4% paraformaldehyde (PFA) and stained with oil-red-O (IHC World, Woodstock, MD) to visualize cytoplasmic lipid-rich vacuoles.
3
Adipogenic Differentiation of Mesenchymal Stem Cells
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Passage 2 MSCs were seeded in six-well plates in triplicate at a final cell density of 5,000 cells per cm2 and propagated in complete medium. Forty-eight hours later, designated as day 0, differentiation was initiated using adipogenic induction medium (AdvanceSTEM adipogenic differentiation medium supplemented with 10% AdvanceSTEM stem cell growth supplement, ThermoScientific, Rockford, IL), as per the manufacturer's instructions. The medium was changed every 3–4 days thereafter, and experiments were terminated after 3 weeks. The differentiated MSCs were fixed with 4% paraformaldehyde (PFA) and stained with oil red O (IHC World, Woodstock, MD) to visualize accumulated cytoplasmic lipid rich vacuoles.24 (link) The lipoid bodies were observed under phase contrast microscopy in at least 10 nonoverlapping fields. To quantify staining, oil red O was extracted with isopropanol containing 4% nonidet P-40 detergent and optical density was then measured at 490 nm.24 (link)
4
Evaluation of Mesenchymal Cell Lineage Differentiation
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For adipogenic and osteogenic differentiation, cells were seeded on to six-well plates that contain differentiation medium. The composition of the differentiation medium is shown in Table 2 . For chondrogenic differentiation, cells were cultured in 5 μl droplets of growth medium in four-well plates for 3 h in the presence of 5% CO2 and changed with chondrogenic differentiation medium plus transforming growth factor β-3 (TGF-β3; Lonza, U.S.A.). All differentiation media were changed every 2–3 days and the differentiation to the three cell lineages was evaluated after 21 days.
To evaluate the differentiation abilities, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and then washed with PBS again. For adipogenic differentiation, accumulation of red lipid vacuoles was observed after Oil Red O staining (IHC World, U.S.A.). For osteogenic differentiation, extracellular calcium deposition was confirmed by Alizarin Red staining (IHC World, U.S.A.). For chondrogenic differentiation, the presence of glycosaminoglycan was verified by Alcian Blue staining (IHC World, U.S.A.). Stained cells were visualized using an inverted microscope.
To evaluate the differentiation abilities, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and then washed with PBS again. For adipogenic differentiation, accumulation of red lipid vacuoles was observed after Oil Red O staining (IHC World, U.S.A.). For osteogenic differentiation, extracellular calcium deposition was confirmed by Alizarin Red staining (IHC World, U.S.A.). For chondrogenic differentiation, the presence of glycosaminoglycan was verified by Alcian Blue staining (IHC World, U.S.A.). Stained cells were visualized using an inverted microscope.
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