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

Published: April 30, 2023

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 Aortic Digestion Solution: 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 solutio…

Representative Results

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 the aortic arch with the brachiocephalic, left common carotid, and left subclavian arteries. Scale bars = 1 mm.

Disclosures

The authors have nothing to disclose.

Materials

15 ml conical tube Falcon 352096
Alpha smooth muscle actin antibody Sigma SAB2500963
Chamber slide Nunc Lab-Tek 154461
Collagenase, Type 2  Worthington LS004176
Dexamethasone Sigma D4902
Dulbecco's Modified Eagle Medium Life Technologies 11965-084
Elastase Sigma E1250
Fetal bovine serum Gibco 16000-044
Forceps, fine point Roboz RS-4972
Forceps, full curve serrated Roboz RS-5138
Formalin (10%) Electron Microscopy Sciences 15740
Hank's Balanced Salt Solution Gibco 14025-092
Insulin syringe with needle Terumo SS30M2913
L-ascorbic acid Sigma A-7506
Micro-dissecting spring scissors (13 mm) Roboz RS-5676
Micro-dissecting spring scissors (3 mm) Roboz RS-5610
Normal Saline Hospira 0409-4888-10
Nuclear fast red Sigma-Aldrich N3020
Penicillin/Streptomycin Corning 30-001-CI
Silver nitrate (5%) Ricca Chemical Company 6828-16
Sodium phosphate dibasic heptahydrate Sigma-Aldrich S-9390
Sodium thiosulfate Sigma S-1648
ß-glycerophosphate disodium salt hydrate Sigma G9422
Tissue culture flask, 25 cm2 Falcon 353108
Tissue culture plate (35 mm x 10 mm) Falcon 353001
Tissue culture plate, six-well Falcon 353046
Trypsin Corning 25-053-CI
Vacuum-driven filtration system Millipore SCGP00525

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Cite This Article
Von Kossa Staining of Aortic Vascular Smooth Muscle Cells: An In Vitro Technique for Assessment of Arterial Medial Layer Calcification. J. Vis. Exp. (Pending Publication), e21033, doi: (2023).

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