Condylar chondrocytes were inoculated in six-well plates at a density of 5 × 105 cells/well. LIPUS stimulation was performed for three consecutive days. After 3 h of LIPUS stimulation on the third day, the cells were fixed with methanol, rinsed 3 times with distilled water and stained with Alcian blue staining solution (Solarbio, China) for 30 min. The staining solution was discarded, and the cells were rinsed, observed and scanned.
Alcian blue staining solution
Manufactured by Solarbio
Sourced in China
Alcian blue staining solution is a laboratory reagent used in histological and cytological applications. It is a cationic dye that binds to acidic polysaccharides, such as glycosaminoglycans, in biological samples. This staining solution can be used to identify the presence and distribution of these acidic macromolecules in tissues or cells.
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Lab products found in correlation
Toluidine blue staining solution, by Solarbio (1 mentions)
Alp kit, by Beyotime (1 mentions)
Alizarin red s, by Solarbio (1 mentions)
Alizarin red staining solution, by Solarbio (1 mentions)
Sodium β glycerophosphate, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
Oil red o staining solution, by Solarbio (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Vitamin c, by Merck Group (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
6 protocols using alcian blue staining solution
1
Alcian Blue Staining of Condylar Chondrocytes
2
Chondrogenic Differentiation of MSCs
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MSCs were cultured in the chondrogenic differentiation medium for 3 days. Subsequently, the medium was removed, and the cells were washed three times with PBS and then stained with toluidine blue staining solution (Solarbio, China) or Alcian blue staining solution (Solarbio, China) for 30 min. Next, the cells were washed with PBS for 5 min, and the washed stained cells were then observed under a microscope.
3
Osteogenic and Chondrogenic Differentiation of BMSCs
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The BMSCs (2 × 104, passage 3) were plated into 12-well plates and cultured in F12 DMEM medium for 12 h, after which the culture medium was changed to osteo-inductive medium (complete medium supplemented with 10 mM β-glycerophosphate, 10 nM dexamethasone, and 50 μM vitamin c). The hydrogels were added and cultured with BMSCs for 7 d. The medium was changed 3 times per week. To evaluate the ability osteogenic differentiation of BMSCs, the alkaline phosphatase (ALP) and mineralization nodule were measured by using ALP kit (Beyotime institute of Biotechnology, China) and alizarin red S (Solarbio, China) staining after then the BMSCs were fixed with 4% paraformaldehyde. For cartilage differentiation, the medium was changed to chondrogenic medium (10 ng/mL transforming growth factor-β3, 100 nM dexamethasone, 50 μM ascorbic acid 2-phosphate and 20 μM l -proline). The subsequent treatment of BMSCs in the 7 d culture was consistent with osteogenesis induction, and then the BMSCs were fixed and stained with Alcian blue staining solution (Solarbio, China). Also, the immunofluorescence staining of collagen II (CoL II) was used to analyze chondrogenic differentiation. All the images were captured with a fluorescence microscopy (LSCM, A1, Nikon, Japan).
4
Induced pluripotent stem cell-derived mesenchymal stem cell differentiation
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For the osteogenic differentiation, the induction medium (IM) included DMEM supplemented with 10% FBS (Gibco, USA), 50 µM vitamin C (Sigma-Aldrich, USA), 10 mM sodium β-glycerophosphate (Sigma-Aldrich, USA), and 0.1 µM dexamethasone (Sigma-Aldrich, USA). iPSC-MSCs were fixed with 95% ethanol after 14 days of differentiation and stained with Alizarin red staining solution (Solarbio, China).
For adipogenic differentiation, cells were cultured in DMEM with 10% FBS, 0.5 µM IBMX (Sigma-Aldrich, USA), 200 µM indomethacin (Sigma-Aldrich, USA), 10 µM insulin (Sigma-Aldrich, USA), and 1 µM dexamethasone. Cells were stained with Oil Red O staining solution (Solarbio, China).
For chondrogenic differentiation, cells were cultured in DMEM with 10% FBS, 10 ng/ml TGF-β1 (R&D Systems, USA), 40 µg/ml proline (Sigma-Aldrich, USA), 100 µg/ml sodium pyruvate (Sigma-Aldrich, USA), 50 µg/ml vitamin C, and 0.1 µM dexamethasone. Cells were stained with Alcian blue staining solution (Solarbio, China).
For adipogenic differentiation, cells were cultured in DMEM with 10% FBS, 0.5 µM IBMX (Sigma-Aldrich, USA), 200 µM indomethacin (Sigma-Aldrich, USA), 10 µM insulin (Sigma-Aldrich, USA), and 1 µM dexamethasone. Cells were stained with Oil Red O staining solution (Solarbio, China).
For chondrogenic differentiation, cells were cultured in DMEM with 10% FBS, 10 ng/ml TGF-β1 (R&D Systems, USA), 40 µg/ml proline (Sigma-Aldrich, USA), 100 µg/ml sodium pyruvate (Sigma-Aldrich, USA), 50 µg/ml vitamin C, and 0.1 µM dexamethasone. Cells were stained with Alcian blue staining solution (Solarbio, China).
5
Chondroitin Sulfate-Induced Mesenchymal Stem Cell Differentiation
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Bone marrow mesenchymal stem cells were seeded onto SF sides containing 4%, 8%, 12% and 16% CS in CM for 14 days. Cells were washed with PBS thrice and fixed with 4% paraformaldehyde. Subsequently, cells were stained with Alcian blue staining solution (Solarbio) and the nuclei were counterstained with redyeing solution. Images were obtained using a microscope (OLYMPUS BX53).
6
Chondrogenic Differentiation of ATDC5 Cells
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Chondrogenic cell line ATDC5 was cultured as previously described.54 (link) Cells were maintained at 37 °C and 5% CO2. DMEM (Gibco, Shanghai, China) with 5% FBS containing 1% Insulin-Transferrin-Selenium (ITS) supplement (Gibco, Shanghai, China) and vitamin C were gently added to induce chondrocyte differentiation for fourteen days. 4 mmol·L−1 chloral hydrate (Sigma-Aldrich. St Louis, MO) was added to cells to remove primary cilia every two days. 10 nmol·L−1 SAG (Selleck, Shanghai, China) was added to cells during chondrogenic differentiation. Cell pellets were fixed with 4% PFA for 15 minutes and stained with Alcian-Blue Staining Solution (Solarbio, Beijing, China) for 30 min to characterize the chondrogenic differentiation.
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