Euthanize the mouse according to your institutional animal care and use committee approved protocol. We anesthetize the mice using a cocktail of ketamine (100 mg/ml) and xylazine (10 mg/ml). The injectable dose recommended is 0.01 ml/g of the body weight of the animal. After injection, allow 10–15 min for the mice to be completely sedated. Check for loss of reflex response by toe pinching or tail pinching. This is necessary to judge the animal’s level of responsiveness to painful stimuli under anesthesia.

Once the animal is sedated, secure the four paws to the surface spreading them as far as possible with the help of adhesive tape. Wet with 70 % ethanol the area where the incision is to be made.

With the help of forceps and scissors, make an incision by cutting the ribs lateral to the sternum to open the thoracic cavity and thus expose the heart.

Perfuse (for frozen sections) or perfuse and fix (for paraffin sections) at a physiological pressure using a gravity flow apparatus (Fig. 1) so as to keep the arteries patent, but not over-distended. Prior to perfusion, flush the tubes of the suspended container with the perfusate (saline) to remove air bubbles. Suspend the perfusate at a height of 60 cm above the mouse maintaining a perfusion pressure of 60–100 mmHg and a flow rate of 1–3 ml/min [5]. This way the arteries are not collapsed or stretched by using excessive or insufficient perfusion pressure, respectively. Insert the 25-G needle attached to the gravity flow apparatus into the left ventricle of the mouse to start perfusion. Make an incision at the right atrium or the hepatic vein. Maintain the flow rate of the perfusate (saline) at 1–3 ml/min so that the pressure is maintained. Perfuse till the liver turns pale in color, which indicates circulating red blood cells have been removed systemically.

Remove the lungs, kidneys, and gastrointestinal and reproductive organs.

Using a dissection microscope, isolate the aortic arch by carefully dissecting the ascending aorta, aortic arch along with BCA, left common carotid artery and left subclavian artery along with 1 mm of descending aorta attached to the aorta. Remove the excess of fat with the help of a cotton swab.

If the aortic root is required, make an incision at the level of the upper level of atria, and cut the heart/aortic root tissue away from the aorta.

Proceed to embedding as outlined below in Subheading 3.2 or 3.3 (see Note 2 ).

Fill separate plastic cryomolds with OCT compound for embedding the heart/aortic tissue and the aortic arch.

With the help of forceps, take the heart/aortic root tissue and embed it in the cryomold containing OCT. Place the axis of the aorta perpendicular to the base of the mold.

For the aortic arch, make an incision between the left common artery and left subclavian artery. Embed the two portions such that the aortic arches are perpendicular to the mold (i.e., they are facing the user).

Freeze the tissues rapidly by placing the molds in dry ice to which isopentane is added.

Cover the tissue molds with aluminum foil and store in −80 °C freezer until cryosections are required.

To section frozen tissue, cut the tissue at 5–6 μm in thickness with a cryostat. Using a thin brush, gently move the curled portion of the section over the cryostat stage. Pick the sections with the help of a brush and place them on poly-l-lysine-coated slides. Poly-l-lysine-coated slides are used to improve tissue adhesion especially since the aortic sections can easily dislodge from the slides during the staining process. For IHC, place 4–5 sections per slide.

Store the cut cryosections in −80 °C freezer immediately. Do not fix the slides before freezing. Fix the slides only if proceeding immediately to immunostaining.

Perform IHC to detect different vascular cells (see below). Although we have used uncut frozen tissues for up to one year, once they are sectioned, they should be further processed within a week if RNA is to be isolated from some of the sections.

Fix the harvested tissues overnight at 4 °C using 4 % paraformaldehyde prepared in PBS or 10 % neutral buffered formalin. Since the aortic tissues are small, overnight tissue fixation is optimal. Usually the fixative volume should be 10× times the tissue volume.

Wash the tissues gently with PBS three times, 5 min each.

Store the tissues in 70 % ethanol till they are ready to be embedded in paraffin blocks.

Transfer tissues to embedding cassettes and dehydrate as follows:

Embed the tissues into paraffin blocks.

To section paraffin blocks, trim and mount the blocks on to the microtome. Take care to trim the paraffin blocks such that there is an optimal cutting surface area that includes the sample with a small paraffin frame.

Using the microtome, cut the tissues in 3–10-μm thick slices. Commonly used thickness for IHC of the mouse aorta is 5–6 μm. Use a brush to gently hold the sections. If the microtome is adjusted properly, the paraffin sections should come out as a ribbon.

Place paraffin ribbons or a slice in 40–45 °C water bath using a second wet brush. As the sections expand on the water bath, the wrinkles on the sections vanish.

Gently scoop out the floating paraffin sections using microscope glass slides and position the section with the help of a brush.

Dry the sections overnight at 37 °C (lower temperatures are better for subsequent antibody detection).

Sections are ready for immunostaining (see Subheading 3.4) or can be stored at room temperature (RT) for an extended period of time.

Bake slides in 56 °C oven for 1 h to melt the wax.

Deparaffinize the tissue sections as follows: