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

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.