sudan iv staining: a procedure for staining lipid-laden atherosclerotic plaques in harvested mouse aortic arch

Sudan IV Staining: A Procedure for Staining Lipid-Laden Atherosclerotic Plaques in Harvested Mouse Aortic Arch

To prepare a pinning bed for en face analysis, fold a segment of paraffin wax film 8 times to make a flat 25 x 25-millimeter surface, and wrap black electric insulation tape around the film to make a dark background for the aorta.
Place a label on the back side of the pinning bed, and use a lead pencil to write down the mouse identification number. Transfer the aortic arch to the pinning bed, and add a drop of PBS to the tissue. Using a stereomicroscope, clean the aorta from the remaining periadventitial adipose tissue, and use Vannas scissors and Dumont forceps to gently peel away all of the surrounding adipose tissue without manipulating or damaging the aorta.
Next, introduce the Vannas scissors into the aortic lumen to expose the intimal surface. Begin cutting the outer curvature of the ascending arch in the distal direction continuing to cut open the branches, including the brachiocephalic artery, and sparing the dorsal part of the descending thoracic region.
To display the intimal surface, cut open the lesser curvature, and fold open the aorta. Using a micro Castroviejo needle holder, secure the open arch to the pinning bed with the blunt end of minutien insect pins without stretching the specimen, gently bending the pins away from the specimen, as the tissue is held in place. Then, store the pinned arch face-down in a Petri dish of PBS at 4 degrees Celsius.
For Sudan IV staining, rinse the specimen for 5 minutes in a Petri dish of 70% ethanol with the arch facing down, before transferring the specimen to a dish of Sudan IV working solution for 7 minutes.
At the end of the incubation, rinse the sample with two 3-minute washes in 80% ethanol to de-stain the normal intimal surface followed by a final rinse in PBS, before returning the tissue to its original Petri dish. Then, acquire micrographs under a stereomicroscope connected to a digital camera at a 10X magnification, obtaining images of the pinned arch submerged in PBS, using small metal weights to hold the pinning bed to the bottom of the Petri dish.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board
1. En Face Analysis of Aortic Arch and Brachiocephalic Artery
<ol>
	<li>Prepare pinning beds for en face analysis of aortic arches. Fold a segment of paraffin wax film eight times to make a flat 25 mm x 25 mm surface. Wrap it with black electric insulation tape to make a dark background for the aorta. Place a label on the backside of the pinning bed and use a lead pencil to write the mouse identification number (normal pen ink will disappear in the staining process).</li>
	<li>Transfer the aortic arch to the pinning bed and place a drop of PBS on top of it. Begin cleaning the aorta from remaining periadventitial adipose tissue under a stereomicroscope.
	<ol>
		<li>Use Vannas scissors and Dumont forceps to gently peel away all surrounding adipose tissue without manipulating or damaging the aorta. The Sudan IV will stain adipose tissue brightly and it is crucial to remove all such tissue at this point. 
		NOTE: Keep the aorta moist at all times applying additional PBS when needed.</li>
	</ol>
	</li>
	<li>Cut open the aorta in the coronal plane by introducing the Vannas scissors in the aortic lumen to expose the intimal surface. Begin to cut the outer curvature of the ascending arch in distal direction and continue to cut open the branches including the brachiocephalic artery. Spare the dorsal part of the descending thoracic region.
	<ol>
		<li>Cut open the lesser curvature and fold open the aorta to display the intimal surface. 
		NOTE: This step requires fine motor skills and needs some practice to master.</li>
	</ol>
	</li>
	<li>Pin the open arch to the pinning bed using the blunt end of minutien insect pins. Use a micro Castroviejo needle holder to put the pins in place. Gently bend the pins away from the specimen when in place. Pin the aorta flat on the bed without stretching the specimen. Store the pinned arch facing downwards in a Petri dish filled with PBS at 4 &#176;C. 
	NOTE: The protocol can be paused here.</li>
	<li>Prepare a working solution of Sudan IV. Mix 1 g of Sudan IV powder, 100 mL of 70% ethanol, and 100 mL of acetone in a dark bottle and gently stir for 10 min. There is no need to filter the solution and it can be used for a couple of months if kept dark at room temperature. If the staining color is not satisfactory, a new solution can be made and the specimens stained again. 
	CAUTION: Acetone is a flammable liquid that could cause serious eye irritation. Store in a well-ventilated place and take precautionary measures when handling.</li>
	<li>Arrange five Petri dishes on the lab bench: one filled with 70% ethanol, one filled with Sudan IV working solution, two filled with 80% ethanol, and one filled with PBS.
	<ol>
		<li>Start with rinsing the specimen in 70% ethanol for 5 min by placing the pinning bed in the first Petri dish with the arch facing downwards. Transfer the specimen to the Sudan IV working solution and let it stain the arch for 7 min.</li>
		<li>Next, rinse in 80% ethanol for 3 min twice to destain the normal intimal surface. Destaining time could be adjusted to optimize results. Lastly, rinse in PBS before putting the specimen back into the original Petri dish.</li>
	</ol>
	</li>
	<li>Acquire micrographs using a stereomicroscope at 10 times magnification connected to a digital camera. Take pictures of the pinned arch submerged in PBS using small metal weights (20 mm x 10 mm x 5 mm) to hold the pinning bed to the bottom of a Petri dish.</li>
</ol>

<table><tbody><tr><td>Black electrical insulation tape (50 mm wide)</td><td>Any specialized retailer</td><td></td><td>To create pinning beds for aortic arches.</td></tr><tr><td>Dissecting scissors (10 cm, straight)</td><td>World Precision Instruments</td><td>14393</td><td>For general dissection of organs.</td></tr><tr><td>Dumont forceps #5 (11 cm, straight)</td><td>World Precision Instruments</td><td>500341</td><td>For microdissection of aorta.</td></tr><tr><td>Ethanol 70% (v/v)</td><td>VWR Chemicals</td><td>83801.29</td><td>Highly flammable liquid and vapour, store in a well-ventilated place, and keep cool.</td></tr><tr><td>Ethanol absolute &amp;ge;99.8%</td><td>VWR Chemicals</td><td>20821.31</td><td>Highly flammable liquid and vapour, store in a well-ventilated place, and keep cool.</td></tr><tr><td>Formaldehyde 4% stabilised, buffered (pH 7.0)</td><td>VWR Chemicals</td><td>9713.1</td><td>Harmful by inhalation, in contact with skin and if swallowed.</td></tr><tr><td>Isopropanol</td><td>Merck</td><td>1096341011</td><td>Flammable liquid, causes serious eye irritation, and may cause drowsiness or dizziness.</td></tr><tr><td>Micro Castroviejo needle holder (9 cm, straight)</td><td>World Precision Instruments</td><td>503376</td><td>For pinning of aortic arches.</td></tr><tr><td>Minutien insect pins, 0.10 mm</td><td>Fine Science Tools</td><td>26002-10</td><td>For pinning of aortic arches.</td></tr><tr><td>Parafilm M</td><td>Bemis</td><td>PM992</td><td>Paraffin wax film used to create pinning beds for aortic arches.</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>-</td><td>-</td><td>Sterile and RNase-free solution is required for perfusion of mice</td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>MZ6</td><td>For dissection and en face documentation</td></tr><tr><td>Sudan IV</td><td>Sigma-Aldrich</td><td>S4261</td><td>Not classified as a hazardous substance or mixture.</td></tr><tr><td>Vannas scissors (8 cm, straight)</td><td>World Precision Instruments&amp;nbsp;</td><td>503378</td><td>For microdissection of aorta.</td></tr></tbody></table>

Source: Centa, M., et al. <a href="https://www.jove.com/v/59828">Quantification of Atherosclerosis in Mice.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure using Sudan IV stain for staining the fatty deposits in the harvested mouse aortic arch to visualize the atherosclerotic plaques and determine the extent of atherosclerosis.

Source: Centa, M., et al. Quantification of Atherosclerosis in Mice. J. Vis. Exp. (2019)
This video demonstrates a procedure using Sudan IV stain for ...

Atherosclerosis occurs due to buildup of lipoproteins in the inner lining of the arteries, resulting in plaque accumulation, thickening and narrowing of the arteries, and reduced blood flow.
Plaques consist of a fibrous cap with smooth muscle cells and extracellular matrix, enclosing a core composed of low-density lipoproteins, LDLs - spherical particles with a hydrophobic core of cholesterol esters and triglycerides, surrounded by a phospholipid outer layer - along with inflammatory cells.
To visualize lipid-rich atherosclerotic plaques, obtain a freshly harvested mouse aortic arch. Transfer it onto a dark background-customized platform for improved contrast during wide-area imaging. Add buffer to prevent tissue dehydration.
Remove the perivascular adipose tissue surrounding the aortic arch to prevent false background staining. Incise the aorta and its branches longitudinally through the lumen to expose the innermost surface with atherosclerotic plaque buildup.
Secure the opened aortic arch onto the platform and incubate in ethanol. Ethanol displaces water from the tissue, inactivating proteins, and preserving the tissue architecture.&#160;
Treat with Sudan IV stain solubilized in organic solvents. Within the plaque, Sudan IV, a fat-soluble diazo dye having high affinity for triglycerides and cholesterol esters of lipoproteins, moves from the organic solvent phase and binds to the LDLs&#8217; hydrophobic core of lipids, forming stable complexes. Wash away excess stain using ethanol.
Under a stereomicroscope, the stained lipid-rich atherosclerotic plaques appear orange-red, while the non-plaque areas appear pale.

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Sudan IV Staining: A Procedure for Staining Lipid-Laden Atherosclerotic Plaques in Harvested Mouse Aortic Arch

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Sudan IV Staining: A Procedure for Staining Lipid-Laden Atherosclerotic Plaques in Harvested Mouse Aortic Arch