The overall goal of this procedure is to prepare the mouse aorta and carotid artery for en face immuno staining after left carotid artery ligation. This method allows us to study organization, each protein expression, and possible state of antral cells and other vascular cells in immuno stain methods. En face preparations allow the analysis of large areas across blood vessels for the assessment of regional difference in the expression of various normal and possible cell parameters.
Demonstrating the procedure will be Dr.Quaico a micro surgeon in our group. Begin by placing the anesthetized mouse onto a surgical board in the supine position. After confirming a lack of response to toe pinch tape the fore paws to the board in the out stretched position and both hind legs to the right side of the mouse to expose to left side of the neck.
Remove the hair around the cervical area and disinfect the exposed skin. Cover the mouse with a sterilized surgical drape with a hole over the surgical region and apply ointment to the animal's eyes. Move the mouse under the dissecting microscope, and make a ventral mid line incision around the cervical area.
Reposition the salivary glands that cover the blood vessels to the left side of the animal to expose the left common carotid artery, which bifurcates into the left internal carotid artery and the left external carotid artery. Gently remove the connective tissue around and below the left internal carotid artery and use a forceps to pass a 2.5 centimeter length of pre cut six O silk suture under the artery, using a second forceps to pull the suture roughly one third of its length to the other side of the vessel. Ligate the left internal carotid artery.
Then remove the connective tissue around the left external carotid artery as just demonstrated, and make a ligation proximal to the left superior thyroid artery. When all of the arteries except the occipital artery have been ligated, return the salivary glands to their original positions and hydrate the surgical field with two to three drops of saline. Then use six O coated vicryl sutures to close the skin and place the mouse in the pre warmed cage with monitoring until full recumbency.
At the termination of the experiment secure the mouse in the supine position on a dissecting board and use iris scissors to expose the abdominal cavity with a mid line incision. Cut the ribs laterally to the sternum to expose the thoracic cavity. Then nick the femoral artery and insert a 26 gauge needle attached to a gravity profusion set up into the apex of the left ventricle to profuse the circulatory system with saline solution supplemented with heparin.
Continue the profusion until the saline flowing from the cut becomes clear. Then switch the profusion system from saline to 4%paraformaldehyde in PBS. After five minutes move the mouse under the dissecting microscope and use blunt end scissors and forceps to harvest the aorta and the left and right carotid arteries.
Next, transfer the tissues into a Petri dish of PBS and carefully remove the fat and connective tissues, followed by the separation, and longitudinal splitting of the aorta and carotid arteries to expose the endothelium. The single most important thing to remember when making en face blood vessel preparations is that the endothelial cells are easily damaged by rough handling. Do not stretch the vessels at any time, especially during the harvesting and cleaning steps.
Then transfer each vessel into individual wells of a 12 well plate containing 0.5 milliliters of permeabilizing solution per well. Permeabilize the blood vessels for 10 minutes with rocking at room temperature, followed by a brief wash in PBS. Block any nonspecific binding with a 30 minute incubation in 10%normal serum from the same species as the planned secondary antibodies in TTBS, with rocking at room temperature.
Then label the samples with the primary antibodies of interest diluted in TTBS with 10%normal serum overnight with rocking at four degrees Celsius. The next morning, rinse the blood vessels with three 10 minute washes in TTBS with rocking at room temperature, followed by incubation in the appropriate secondary antibodies diluted in TTBS and 10%normal serum and dappy. Return the samples to the rocker and cover to protect from light.
After one hour at room temperature with rocking wash the samples three times in fresh TTBS as demonstrated, followed by a brief rinse in PBS, and place one drop of anti fade reagent per vessel onto individual 22 by 50 millimeter cover glasses. Under a microscope, place one vessel into each drop of reagent with the endothelium facing down, and cover the vessels with one 22 by 75 millimeter slide glass per sample. Place the slides on a clean laboratory wipe and cover them with two pieces of laboratory wipe and 3.5 kilograms of weight to flatten the vessels.
After a maximum of five minutes, remove the weight, and wipe the excess solution from around the cover slips. Then apply nail polish to the four corners of the cover slips and place the slides in a slide box, cover slip side up. The next morning use more nail polish to seal the cover slips completely and image the vessels as soon as the nail polish is dry.
Here a typical en face immuno florescence image of endothelial double stained with anti VE cadherin and anti vascular cell adhesion molecule one antibodies and illustrating a single optical section of a mouse aorta taken near the opening of an intercostal artery is shown. Note the green linear outline staining at the adherence junction of the endothelial cells and the strong anti vascular cell adhesion molecule one staining at the opening of the intercostal artery where the disturbed blood flow is known to occur. In these images the en face staining of a partially ligated left carotid and a control unligated right artery one day after the surgery can be observed, with an increased anti vascular cell adhesion molecule one staining apparent in the ligated vessel.
After watching this video you should have a good understanding of how to make a partial left carotid artery ligation and to make en face preparations in mouse blood vessels. Take this with your optical to other small animals. The use of en face preparations is now limited to immuno florescence staining, but can also be used with other methods, such as for study of those whole regions after oriole staining, or for ex vivo counter experiments.
