Whole-mount Alcian blue staining to visualize the cartilage was performed according to the previous methods (Walker and Kimmel, 2007 (link)). In brief, zebrafish larvae were maintained in 0.2 mM PTU from one dpf, fixed with 4% PFA overnight at 4°C, dehydrated with 50% ethanol for 10 min, stained with acid-free stain solution (0.02% Alcian blue (A5268, Sigma-Aldrich), 60 mM MgCl2, 70% ethanol) at room temperature overnight, cleared with 50% glycerol and 0.25% KOH at room temperature for 2 hr, dipped into 50% glycerol and 0.1% KOH, and viewed and photographed with a stereoscopic microscope (SZX10, Olympus).
A5268
Manufactured by Merck Group
Sourced in United Kingdom
The A5268 is a laboratory equipment product. It is a device used for specific functions in a laboratory setting. No further details about its core function or intended use are provided.
Automatically generated - may contain errors
5 protocols using a5268
1
Visualizing Cartilage Development in Zebrafish
2
Skeletal Staining and Imaging Protocol
3
Skeletal Staining and Imaging Protocol
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Whole mount skeletons were stained with 0.05% alizarin red (A5533, Sigma-Aldrich) and 0.15% alcian blue 8GX (A5268, Sigma-Aldrich), and then transferred with glycerol/KOH solutions from 20% glycerol/1% KOH to 80% glycerol/1% KOH. For whole-mount lacZ staining, reporter mice were fixed in 1% formaldehyde and 0.2% glutaraldehyde in PBS for 90 min, stained in X-gal for 2 h at 37°C, and cleared in 80% glycerol/PBS. For histological analysis with alcian blue and von Kossa staining, sections were stained with 1% aqueous silver nitrate (196-00831, Fujifilm) solution and 1% alcian blue (pH 2.5, 8GX, Sigma-Aldrich) solutions. Unreacted silver was removed using 5% sodium thiosulfate. Counterstaining was performed using nuclear fast red staining. Micro-computed tomography (μCT) scanning of the harvested femurs was performed using a microfocus X-ray CT system SMX-90CT (Shimadzu, Kyoto, Japan); the three-dimensional construction software package TRI/3D-BON (Ratoc System Engineering, Tokyo) was used for quantitative analysis.
4
Alcian Blue Staining of Tissue Sections
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Tissue sections were rehydrated (as described above) and stained for 15 min in Alcian blue solution (1% Alcian Blue in 3% solution of acetic acid) (Cat. # A5268, Sigma Aldrich, UK). Slides were then washed in running tap water for 2 min. Nuclei were stained in Haematoxylin solution for 1 min and sections were then washed in tap water for 2 min. Slides were finally dehydrated (as described before), cleared and mounted in DPX.
5
Tissue Staining with Alcian Blue and Eosin
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Sections were post fixed in 4% PFA for 5 min at room temperature and then washed with distill water. 0.1% Alcian blue (Sigma #A5268)/0.1 M HCL (Sigma # H1758) solution was applied for 3 min at room temperature, followed by staining in 0.02% eosin for 2 min.
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