This article discusses the role of vascular smooth muscle cells (VSMCs) in vascular calcification, a process influenced by pathological cues and aging. It details a staining method to visualize calcification in VSMCs using von Kossa staining.
Vascular smooth muscle cells, or VSMCs, are the prominent cellular components in the tunica media - the middle layer of the blood vessel wall. In response to pathological cues or aging, these cells mediate vascular calcification - a phenomenon of calcium accumulation in the extracellular matrix.
Take a multi-well plate containing an adherent layer of pre-calcified, murine aortic VSMC culture. Incubate the cells with formalin solution to cross-link cellular proteins, and stabilize the VSMC structure before staining.
Begin the von Kossa staining by aspirating formalin from the well, and adding silver nitrate solution. Incubate the plate under an appropriate light source.
Once the silver nitrate solution reaches the calcification sites in the extracellular matrix of the VSMC layer, the silver ions replace the calcium ions bound to phosphate. This results in a transient yellow coloration. In the presence of organic matter and light, the photochemical degradation of silver phosphate results in black-colored metallic silver formation.
Discard the maximum amount of spent silver nitrate solution. Treat the cells with sodium thiosulfate solution. This solution converts residual silver salts into a water-soluble complex, which can be removed via a final rinse with water, improving staining contrast.
Image the stained cells to locate the insoluble black deposits indicating 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.
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