2.13. Von Kossa Staining of Calcium Deposition by Osteoblasts after ADSC-EV Treatment

Calcium that was deposited in the calcified matrix by osteoblasts was visualized using von Kossa staining [45]. At specific time points, the hFOBs and hOBs were washed twice with PBS and fixed using 10% formalin for 30 min. The cells were incubated in 5% silver nitrate (Sigma-Aldrich, St. Louis, MO, USA) under a lamp for 20 min until the calcium turned black; after which, the cells were washed with 5% sodium thiosulfate for 5 min. Through the aforementioned staining method, the mineral matrix was stained black. Images were captured using a camera.