The red alizarin assay was used to evaluate the calcium deposition in OS cell lines after treatment for 48 h with RSV (60 and 120 µM). The OS cell lines were seeded to 1.5 × 104 cells/well in 12-wells plates, then treated with RSV. After this time, every well was washed with PBS and fixed using 4% PFA at room temperature for 30 min. Double-distilled water (ddH2O) was used to rinse the plates twice; the plates were incubated with 2% alizarin red S (ARS) staining solution (Sigma-Aldrich) at room temperature for about 30 min. Finally, cells were gently washed by ddH2O, and images were observed via a light microscope (Nikon Eclipse TI-S) [71 (link)].
Alizarin red s staining solution
Manufactured by Merck Group
Sourced in United States
Alizarin red S staining solution is a laboratory reagent used for the histological staining of calcium deposits and mineralized tissues. It is an anthraquinone dye that binds to calcium ions, producing a bright red color. The solution is typically used in microscopy techniques to visualize and analyze the presence and distribution of calcium-containing structures within biological samples.
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Lab products found in correlation
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
Eclipse ti s, by Nikon (1 mentions)
Mvx10 macroview, by Olympus (1 mentions)
Calcium colorimetric assay kit, by Nanjing Jiancheng (1 mentions)
Sodium thiosulphate, by Merck Group (1 mentions)
Imt 2, by Olympus (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Eos 70d, by Canon (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
Alp kit, by Merck Group (1 mentions)
Bcip nbt substrate solution, by Merck Group (1 mentions)
Silver nitrate, by StatLab (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Osteogenesis quantitation kit, by Merck Group (1 mentions)
Leica sp1600, by Leica Biosystems (1 mentions)
Biorevo bz 9000, by Keyence (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Gryphax microscope camera, by Jenoptik (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Metformin, by Targetmol (1 mentions)
23 protocols using alizarin red s staining solution
1
Alizarin Red S Assay for Calcium Deposition
2
Extracellular Matrix Mineralization in BMSCs
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12 days after culturing under NP, the extracellular matrix mineralization of BMSCs were evaluated by the alizarin red S staining and calcium contents. The BMSCs were fixed in 4% paraformaldehyde for 15 min and gently rinsed with PBS. Finally, the sample were then incubated with 40 mM alizarin red S (ARS) staining solution (Sigma) for 30 min at room temperature. Images were recorded with an Olympus MVX10 MacroView (Japan). For quantitative analysis, calcium contents were measured using a calcium colorimetric assay kit (Nanjing Jiancheng, China) according to the manufacturer’s instructions. All samples were examined at less three times.
3
Quantifying ADSC Mineralization with ARS
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According to a previous study [25 (link)], ADSCs were cultured in 35-mm plates for 14 days. After fixation in 95% ethanol, the ADSCs were stained with 1% Alizarin red S (ARS) staining solution (Sigma‐Aldrich) at room temperature for 15 min. To quantify the mineralization levels, densitometric analysis of staining was performed using ImageJ 1.48.
4
Mineral Deposition Analysis by ARS and von Kossa
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Mineral deposition was examined by alizarin red S (ARS) and von Kossa staining. PDLPs were cultured and treated as described in the ALP activity assay. On day 21, cells were washed with PBS twice, fixed for 30 min using 4% paraformaldehyde. For ARS staining, cells were stained using 2% alizarin red S staining solution (Sigma-Aldrich) for 5 min. The unbound ARS were washed with water. For von Kossa staining, the fixed cells were washed with deionized water and incubated with 5% silver nitrate (Sigma-Aldrich) solution for 1 h under ultraviolet light, followed by treatment with 10% sodium thiosulphate (Sigma-Aldrich) for 5 min. As for both staining methods, the plates were examined using an inverted optical microscopy (Olympus IMT-2, Tokyo, Japan) and photographed using a digital camera (Canon EOS 70D, USA). ARS was quantified by measuring the absorbance at 562 nm after the stained cells were desorbed with 10% (w/v) cetylpyridinium chloride (Sigma-Aldrich). The von Kossa staining was quantified densitometrically with Image J.
5
Melatonin's Impact on Osteogenic Calcification
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To examine the potential effect of melatonin on osteogenic calcification activity, RAOECs were washed with PBS and fixed with 4% paraformaldehyde for 30 min after osteogenesis induction. ALP staining to detect osteogenesis was performed with cells cultured in osteogenic medium for 14 days according to ALP kit (Sigma-Aldrich). For Alizarin red staining, the cells were cultured for 21 days and then stained with 2% Alizarin red S staining solution (pH 4.2; Sigma-Aldrich) for 30 min at 37°C to visualize matrix calcification in the culture medium.
6
Osteogenic Differentiation of ASCs
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ASCs at passage 4 were cultured at a seeding density of 1 x 105/well in a six-well plate. The ASCs were maintained in MesenPRO RS Medium until the cells reached 80% confluence. The StemPro Osteogenesis Differentiation Kit (Gibco, USA) was used to induce the ASCs to differentiate into osteoblasts. The cells were maintained in osteogenic differentiation medium for 21 days, during which the medium was changed every 3 days. When the differentiated cells were removed from the incubator after 21 days, the differentiation was confirmed by Alizarin Red S staining and alkaline phosphatase (AP) staining. For the Alizarin Red S staining, the differentiated cells were washed with DPBS, fixed with 10% neutral buffered formalin for 1 hr, stained with Alizarin Red S staining solution (Millipore, USA) in the dark for 45 min at RT and analyzed using an inverted phase-contrast microscope. For the AP staining, the differentiated cells were washed with DPBS and fixed with 10% formalin for less than 1 min because a longer incubation would inactivate AP. The cells were then stained with 5-bromo-4-chloro-3-indolyl-phosphate with nitro blue tetrazolium (BCIP/NBT) substrate solution (Sigma-Aldrich, USA) at RT in the dark for 5-10 min and washed with DI water, after which images were observed using an inverted microscope.
7
Histochemical Staining Techniques for Cell and Tissue Analysis
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For alizarin red S staining, the cells were fixed with 10 % formalin for 20 min and then incubated with alizarin red S staining solution (Millipore) for 20 min. For Von Kossa staining, cells were fixed with 10 % formalin for 20 min and then exposed to ultraviolet light in 5 % silver nitrate (American Master Tech, Lodi, CA, USA) for 1 h. After washing with DW, the cells were incubated in 5 % sodium thiosulfate (American Master Tech) at room temperature for 3 min and then observed under an inverted microscope (Olympus, Tokyo, Japan). For Alcian blue staining, chondrogenic spheroids were fixed with 10 % formalin for 30 min and embedded in 2 % agarose (LPS solution, Seoul, Korea) in PBS. Sections of chondrogenic spheroids were treated with 3 % acetic acid (Millipore) for 3 min and then incubated in Alcian blue staining solution (American Master Tech) for 30 min. The stains were detected using an inverted microscope.
8
Osteoblast Mineralization Quantification
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The implants were disposed on specific supports or scaffolds to allow the cellular culture of pre-osteoblasts (MC3T3-E1) on their surfaces. Cells were seeded at a density of 1 × 105 cells/scaffold, kept for 28 days and maintained under cell culture conditions. The α-MEM medium was changed every 3 days and bone mineralization inducers (β-glycerol phosphate, acid ascorbic and melatonin) were added to the positive control. After this period, cells were fixed and the mineralization process was revealed using Alizarin Red S staining solution (40 mM pH 4.2 for 1 h) to detect the calcium matrix (osteogenesis quantitation kit – Millipore, Burlington, VT, USA). Images of the mineralized matrix were acquired by digital microscopy with high resolution LED (KH 7700–Hirox-Europe, Tokyo, Japan). Undifferentiated osteoblasts without extracellular calcium deposits were slightly reddish, whereas mineralized osteoblasts with extracellular calcium deposits resulted in a bright orange-red color.
9
Alizarin Red S Staining for MSC Mineralization
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Alizarin Red S staining was performed to assess mineralization of MSCs on SPAs, TPAs and BPXs. After 7 and 14 days of cultivation MSC-seeded constructs were fixed in 4% PFA for 1 hour, washed thrice with PBS and consecutively incubated in 500 μL Alizarin Red S staining solution (40 mM, Merck, Darmstadt, Germany) for 60 min at room temperature on an orbital shaker. After washing MSC-seeded constructs with distilled water Alizarin Red S was extracted using a 10% (w/v) cetylpyridinium chloride (CPC) solution (Carl Roth, Karlsruhe, Germany) for 48 hours. Finally, Alizarin Red S was quantified by measuring the optical density at 560 nm and calculated in accordance with the standard curve. At each time point Alizarin Red S quantification was performed for two independent grafts and technical replicates for 3 donors.
10
Quantitative Analysis of Embedded ET
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Due to the height of the block with the embedded ET, it had to be divided into two blocks of half the height for cutting with the hole saw (Leica—SP1600®, Leica Biosystems Nussloch GmbH, Nussloch, Germany). Cone-beam computed tomography (CBCT) was performed with both halves (XORAN xCAT®, Xoran technologies, MI, USA, ENT scan, high-resolution 0.3 mm). Subsequently, 100 sections of 33 μm thickness were produced with the hole saw at equal distances of 330 μm (the thickness of the sawing blade). After each section, the remaining thickness of the respective block was measured at the three grooves. Two sections were lost due to mounting of the blocks on the sample holder (Fig 1a ). Sections were stained with methylene blue (Loeffler’s Methylene blue solution; Merck) for 45 s at 80 °C and alizarin red (Alizarin red S staining solution; Merck) for 1.5 min. Digitization of the sections was performed using a digital microscope (Biorevo BZ-9000®, KEYENCE, Osaka, Japan) at 2x magnification and a resolution of 3094 x 4094 pixels.
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