ARS-specific dye with affinity to calcium ions was used as an indicator for “Ca2+” deposition in the mineralized matrix. Fixed cells in 4% PFA were washed in distilled water three times at 5 min interval. The cells were incubated with 1% ARS staining solution (pH 4.2, Roth, Germany) at room temperature for 30 min. Excess of staining was washed three times in distilled water for 5 min to remove the unbound dye. The morphological alterations following each time point were examined and were photographed using a light microscope equipped with a digital camera and the LAS V4. 4 Software (Leica, Germany).
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