Following viability assay, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then diluted down to 1% PFA using PBS for long term storage. Proteoglycan formation was analyzed using an alcian blue staining kit (ScienCell, Carlsbad, CA). The wells were washed with 0.1 N HCl and then covered with alcian blue overnight at room temperature. The wells were then washed with ultrapure water 3 times prior to dye extraction using 6 M guanidine HCl overnight at room temperature. The extracted dye was measured using a microplate reader (DTX880 Multimode Detector, Beckman Coulter) at 630 nm. A standard curve of alcian blue values from 635 to 9.76 μg/mL was used for quantification.
Alcian blue staining kit
Manufactured by ScienCell
Sourced in United States
The Alcian blue staining kit is a laboratory tool used for the detection and visualization of acidic polysaccharides, such as glycosaminoglycans, in tissue samples. The kit provides the necessary reagents and protocols for performing Alcian blue staining, a commonly used histochemical technique.
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4 protocols using alcian blue staining kit
1
Alcian Blue Staining of Proteoglycans
2
Quantitative Analysis of Proteoglycan Formation
3
Detailed Staining and Analysis Protocol
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After 10 days of culture, cells were collected and stained. For alizarin red staining and quantitative analysis, the cells were rinsed with PBS, fixed in 4% paraformaldehyde for 15 min, washed with ddH2O three times, and then stained with Alizarin Red S Kit (Solarbio, China) for mineralized nodules. The cells were washed again before taking photos. After that, 10% cetylpyridinium chloride was used to dissolve the nodules, and the OD (optical density) was measured at 562 nm. For ALP staining and activity analysis, cells were rinsed with PBS and treated with 1% Triton X-100. The ALP activity of the cell lysates was tested with an ALP Activity Assay Kit (Beyotime, China). The protein concentrations were examined using an Enhanced BCA Protein Assay Kit (Beyotime) following the manufacturer’s instructions. For alcian blue staining and analysis, cells were also fixed in 4% paraformaldehyde for 10 min and then washed and stained with an Alcian Blue Staining Kit (ScienceCell, USA). After staining, the cells were washed with diH2O 3 times, and 6 mol/L guanidine hydrochloride was added and incubated for 2 h at room temperature. The OD was measured at 630 nm, and the cells were observed and captured under an inverted microscope (Olympus IX73, Japan).
4
Alcian Blue Staining of Slides
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Slides samples were hydrated with PBS, then stained with Alcian blue according to Alcian Blue Staining Kit instructions (ABlue, ScienCell Research Laboratories, Carlsbad, CA, USA).
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