Alizarin red staining to determine mineralized nodules

A 1% alizarin red aqueous solution was created by dissolving1 g Alizarin Red powder (Shanghai Yuanye Biological Co., Ltd, China) in 100 mL distilled water. This was filtered to remove impurities, the pH was adjusted to 4.2 with 10% ammonia water, and the solution was then stored at 4 °C until use. The Osteoblasts were cultured in a 12-well plate, then the medium was discarded, PBS was used to rinse the osteoblasts, 0.5 mL of 95% ethanol was added, and they were fixed for 30 min, or overnight, at 4 °C. After fixation, the ethanol was discarded and a little air-drying was allowed. Next, 0.3 mL of the prepared Alizarin Red S staining solution was added to each well for 15 min, the time of which can also be extended appropriately according to the staining situation. The staining solution was vacuumed off (recyclable), followed with rinsing in distilled water 3 times. The cells were then routinely dehydrated, mounted, observed, and photographed under a light microscope (Guangzhou Minghui Technology Co., Ltd., China).