The hDPSCs were differentiated into chondrocytes using chondrogenic differentiation medium and E. faecium L-15 extract for two weeks. Pellets were fixed with 4% paraformaldehyde for 30 min. Pellets were washed with phosphate-buffered saline (PBS) and incubated with alcian blue staining solution (Merck, Darmstadt, Germany) in the dark for 1 h at room temperature. Pellets were rinsed three times with ddH2O to neutralize the acidity. For quantitative analyses, alcian blue-stained cells were dissolved in 6 M guanidine hydrochloride (Sigma–Aldrich) for 6 h. The absorbance of the solubilized solution was measured at 650 nm.
Alcian blue staining solution
Manufactured by Merck Group
Sourced in United States, Germany
Alcian blue staining solution is a laboratory reagent used for the histological detection and visualization of acidic polysaccharides, such as glycosaminoglycans, in biological samples. It provides a blue staining of these compounds, allowing their identification and localization within tissue sections or cell preparations.
Automatically generated - may contain errors
Lab products found in correlation
Alcian blue, by Merck Group (2 mentions)
Oil red o, by Merck Group (2 mentions)
Alizarin red s, by Merck Group (2 mentions)
Inverted microscope, by Leica (2 mentions)
6 m guanidine hydrochloride, by Merck Group (1 mentions)
Luminescent atp detection assay kit, by Abcam (1 mentions)
Dextran sulfate sodium salt colitis grade, by MP Biomedicals (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Stempro chondrogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Stempro differentiation kit, by Thermo Fisher Scientific (1 mentions)
Alizarin red s solution, by Merck Group (1 mentions)
Oil red o staining solution, by Merck Group (1 mentions)
Buffered formalin, by Thermo Fisher Scientific (1 mentions)
Safranin o, by Merck Group (1 mentions)
Histo clear 2, by National Diagnostics (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Bcip nbt kit, by Merck Group (1 mentions)
Stempro differentiation media, by Thermo Fisher Scientific (1 mentions)
Oil red o kit, by Merck Group (1 mentions)
15 protocols using alcian blue staining solution
1
Chondrogenic Differentiation and Alcian Blue Staining
2
Colorectal Cancer Molecular Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Abcam (Cambridge, MA) was the source of the luminescent ATP detection assay kit (ab113849). Isolation of genomic DNA from tail biopsies and tumor samples was achieved with the DNeasy Blood & Tissue Kit (69506) purchased from Qiagen (Valencia, CA). Applied Biosystems TaqMan®MGB (minor groove binder) probes with a 5´ reporter (FAM or VIC) and a 3´ nonfluorescent quencher (MGB-NFQ), Invitrogen™TRIzol™ Reagent (15596026), Invitrogen SuperScript™Vilo™cDNA Synthesis Kit (11754050), and SequalPrep™ Long PCR Kit with dNTPs (A10498) were purchased from ThermoFisher Scientific (Waltham, MA). Midland Certified Reagent Company (Midland, TX) synthesized the PCR primers. OmniPur agarose (2120) was purchased from Calbiochem division of EMD4Biosciences (San Diego, CA). Promega (Madison, WI) was the source of the Lambda DNA/EcoRI + HindIII agarose gel markers (G173A). Azoxymethane (A2853) was purchased from Sigma-Aldrich (St. Louis, MO) and dextran sulfate sodium salt colitis grade (160110) was purchased from MP Biomedicals (Santa Ana, CA). Mouse Mitochondrial DNA copy number assay kit (MCN 3) was purchased from Detroit R&D, Inc. (Detroit, MI). Alcian-blue staining solution was purchased from EMD Millipore Corp (Burlington, MA). All protocols are accessible in protocols.io (dx.doi.org/10.17504/protocols.io.4argsd6 ).
3
Alcian Blue Staining of Differentiated MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After chondrogenic differentiation, the MSCs were washed with PBS, fixed with paraformaldehyde (4%), washed and incubated for 10 min with 0.1 m HCl. Then samples were incubated overnight with alcian blue staining solution (Merck, Darmstadt, Germany), then washed with aqua dest. and analysed using a Zeiss Axiovert 200 microscope.
4
Chondrogenic Differentiation of ASCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 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 Chondrogenesis Differentiation Kit (Gibco, USA) was used to induce the ASCs to differentiate into cartilage spheroids. The ASCs were maintained in the chondrogenesis differentiation medium for 14 days, during which the medium was changed every 3 days. When the differentiated cells were removed from the incubator after 14 days, they were washed with DPBS. The cells were then fixed with 10% formalin for 1 hr. After that, the cells were washed with DI water and stained with 2 ml of Alcian-Blue staining solution (Millipore, USA) overnight at RT in the dark. On the subsequent day, the staining solution was removed, and the cells were washed with destaining solution (ethanol (98-100%): acetic acid (98-100%) at a dilution ratio of 3:2) for 20 min. The destaining solution was removed, and DPBS was added to cover the cell monolayer, after which images were observed using an inverted phase-contrast microscope.
5
Multilineage Differentiation Assay for Cardiac Stem Cells
6
Alcian Blue Chondrogenic Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded and treated as described above. The cells were washed with PBS and fixed with 10% neutral buffered formalin for 60 min at room temperature. After two rounds of washing with distilled water, the cells were incubated overnight in the dark at room temperature with Alcian blue staining solution (Sigma-Aldrich, St. Louis, Missouri, USA). Heterogeneous chondrogenic differentiation was identified as intense dark-blue coloration due to the formation of aggrecan.
7
Multilineage Differentiation Assay of TMSC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the mesodermal differentiation capacity of TMSC, TMSC (5 x 104 cells) were seeded in 24-well cell culture plates. After 24 h of seeding, the cells were incubated in commercially available adipogenic, osteogenic and chondrogenic differentiation media (Invitrogen) as previously described [19 (link)]. The differentiation media was changed every 3 or 4 days for 2 weeks. After induction of differentiation, the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and washed again with PBS. Thereafter, cells were stained with 2% Oil red O staining solution (Sigma-Aldrich) for adipogenic differentiation, with 2% Alizarin red S solution (Sigma-Aldrich) for osteogenic differentiation, or 1% Alcian blue staining solution (Sigma-Aldrich) for chondrogenic differentiation. After the cells were rinsed, the adipogenic, osteogenic and chondrogenic cells were visualized under a phase-contrast microscope.
8
Histological Staining of Paraffin-Embedded Samples
9
Trilineage Differentiation of hUC-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For all differentiation assays, hUC-MSCs were first cultured in microcryogels for 0, 3, and 5 d before being digested, and were subsequently seeded in six-well plates. Cells were cultured in growth medium for 12 h followed by changing to differentiation medium. The trilineage differentiation ability of hUC-MSCs was assessed by culturing the cells in StemPro® differentiation media (Gibco, Life Technologies, USA) to induce osteogenesis, chondrogenesis, and adipogenesis. Osteogenesis was assessed at 14 d by staining the cells for alkaline phosphatase with the BCIP/NBT Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Chondrogenesis was assessed at 14 d by staining for sulfated proteoglycan deposits with Alcian-Blue Staining Solution (Sigma-Aldrich). Adipogenesis was assessed at 21 d by staining for lipid accumulation with an Oil Red O Kit (Sigma-Aldrich).
10
Isolation and Characterization of hUMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hUMSCs were extracted using the tissue block culture attachment method, as previously reported [19 (link),20 (link)]. The preparation of P5 hUMSCs was conducted in a GMP facility following good clinical practice (GCP) guidelines. The identification of hUMSCs was detected by flow cytometry (BD Biosciences, FACSAria2, USA). The antibodies used in this experiment included CD73-BV421 (cat.562430, BD Biosciences, USA), CD105-APC (cat. 562408, BD Biosciences, USA), CD90-FITC (cat.555595, BD Biosciences, USA), HLA-DR-PE (cat. 555812, BD Biosciences, USA), CD34-PerCP-Cy™5.5 (cat. 347203; BD Biosciences, USA), and CD45-FITC (cat.555482; BD Biosciences, USA). Osteogenic differentiation medium (catalog#05465, STEMCELL Technologies, China), adipogenic differentiation medium (catalog#05412, STEMCELL Technologies, China), and chondrogenic differentiation medium (catalog#05455, STEMCELL Technologies, China) were used to assess the osteogenic, adipogenic, and chondrogenic differentiation potential of hUMSCs, following the manufacturer’s instructions. Oil Red O solution (O1391, Sigma-Aldrich, USA), Alizarin Red S (A5533, Sigma-Aldrich, USA), and Alcian Blue staining solution (TMS-010-C, Sigma-Aldrich, USA) were used for adipogenic, osteogenic, and chondrogenic staining, respectively, according to the manufacturer’s instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!