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

In This Article

Abstract

Source: O'Rourke, C., et al. Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation. J. Vis. Exp. (2016).

This video demonstrates a staining technique for assessing arterial medial calcification of the vascular smooth muscle cells in vitro. The von Kossa method employs a photochemical reaction to deposit metallic silver at the sites of calcification and impart a black coloration.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Reagents

Isolation and Calcification of Murine Aortic VSMCs
1. Aortic Digestion Solution:
  1. Prepare a fresh solution (~3-5 ml per aorta harvested) with Hank's Balanced Salt Solution (HBSS) containing 175 U/ml type 2 collagenase and 1.25 U/ml elastase. Sterilize the solution with a 0.22 µm vacuum-driven filtration system and keep the solution on ice until use.
2. Cell Media:
  1. Supplement 500....

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Results

Anatomy diagram of pig arteries labeled; features heart, aorta, carotid, iliac artery.
Figure 1: Representative Dissection of an Aorta from a Wild-type Mouse. (A) A picture of the thoracic and abdominal aorta extending to the common iliac artery bifurcation is depicted after removal of the overlying organs and peri-aortic fat. (B) A focused view of th.......