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 |
Tags
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).