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

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

1. 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).
2. 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.
   1. 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.
3. 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.
   1. 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.
4. 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 °C.
   NOTE: The protocol can be paused here.
5. 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.
6. 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.
   1. 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.
   2. 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.
7. 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.