Histologic studies were performed on scaffold cultures at 24 days and 6 weeks. Scaffolds were fixed at 10% normal buffered formalin, embedded in paraffin, and sectioned at 4 microns using standard techniques. The sections were deparaffinized and stained with hematoxylin and eosin, Alizarin Red, or anti-p-Smad1/5 (Cell Signaling Technologies, Beverly, MA) and processed with the Dako automated FLEX system (Dako, Carpinteria, CA). All slides were analyzed qualitatively using a standard microscope and digitally photographed at 20× magnification.
Alizarin red
Manufactured by Cell Signaling Technology
Alizarin Red is a laboratory dye used for the detection and visualization of calcium deposits in biological samples. It binds to calcium ions, forming a red-colored complex that can be observed using microscopy or spectrophotometric techniques.
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2 protocols using alizarin red
1
Histological Analysis of Scaffold Cultures
2
Quantitative Histological Analysis of Scaffold Mineralization
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Histology was performed on scaffolds at 0, 3, 7 days of culture. Scaffolds were fixed in 10% formalin, paraffin-embedded, and sectioned at 4 µm. Following deparaffinization, the sections were stained with Alizarin Red at 10 mg/ml (Cell Signaling Technologies, Beverly, MA). Slides were analyzed qualitatively using a standard microscope and digitally photographed. For quantification, add 10% glacial acetic acid (Fisher Scientific International, Inc., Pittsburgh, MA) to the staining sections for 30 minutes. Absorbance of the incubation medium was measured at 450 mm (Epoch spectrophotometer, BioTek, Winooski, VT).
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