Von Kossa Staining of Aortic Vascular Smooth Muscle Cells: An In Vitro Technique for Assessment of Arterial Medial Layer Calcification

To induce calcification in vascular smooth muscle cells, transfer the cells to the six-welled culture plate, and allow them to adhere. Next, re-feed the cells in calcification media. Incubate the cells at 37 degrees Celsius for at least seven days. Afterward, aspirate the media from the cell culture plate. Wash the cells once in deionized water.

Fix the cells by placing them in 1 milliliter of 10% formalin at room temperature for 20 minutes. Then, remove the formalin and wash the fixed cells with distilled water for 5 minutes.

Next, incubate the cells in 1 milliliter of 5% silver nitrate solution under a 60 to 100-watt bulb for 1 to 2 hours. Afterward, aspirate the silver nitrate solution, and wash with distilled water for 5 minutes. After 5 minutes, remove the unreacted silver by placing the cells in 1 milliliter of 5% sodium thiosulfate solution in distilled water for 5 minutes. Subsequently, rinse the cells with distilled water for 5 minutes. The cells stained with von Kossa are now ready for imaging with standard inverted light microscopy.