Anesthesia should be induced at ∼3% isoflurane in a carrier gas flowing at 1 L/min (medical grade air, or 70/30 mixture of N₂O/O₂ will work but are listed here in order of preference).

Upon induction, place animal on the heating pad ventral surface up with its nose in a nose cone to continue to deliver isoflurane at ∼1.5–2% in 1 L/min of the carrier gas. The animal’s head should be pointing away from you.

Before proceeding, check for the appropriate plane of anesthesia (e.g., by pinching one of the hindpaws of the animal).

Opthalmic ointment should be placed on each eye.

The fur over the left femoral artery at the level of the inguinal ligament should be removed by using clippers or depilatory (see Note 10)—externally the landmark is where the hindlimb attaches to the intraperitoneal (IP) cavity (Fig. 2).

Prepare the skin over the femoral artery by scrubbing it with three separate povidone–iodine swabsticks followed by one to two alcohol pads. Let the skin dry.

At this point, transfer the animal to the surgery area and put on sterile gloves. Place the sterile drape over the animal so that the surgical aperture is over the furless skin.

Using the large, delicate serration forceps and small surgical scissors, make a small incision (∼1 cm in length) through the skin, only, over the femoral artery at the level of the inguinal ligament. The incision should be parallel to the inguinal ligament (see Fig. 2).

You will need to move aside a small portion of subcutaneous fat before visualizing the femoral artery, which will reside between the femoral vein on the left and the femoral nerve on the right.

Have a small syringe (1–3 mL) filled with sterile saline available to moisten the surgical site as you are separating the femoral artery from surrounding tissue.

Isolate the femoral artery from the vein using the small, angled, smooth platform forceps (see Note 11).

When the femoral artery has been isolated, run a 5-0 silk suture beneath it and tie off the artery. A drop or two of saline can provide lubrication. Cut the ends of the ligature to appropriate lengths (∼2 mm) (see Note 12).

Close the skin using the needle holders, 5-0 silk suture, and a wound clip. The wound clip should be removed after 7 days if it is still in place.

Administer appropriate post-operative analgesic, e.g., buprenorphine (0.05–0.1 mg/kg subcutaneously).

Recover the animal in a cage, with no bedding, that is placed half-on/half-off a warm heating pad until ambulatory.

Gently restrain animal by holding the skin at the nape of the neck between thumb and forefinger with one hand and tail with the other hand.

Turn animal so its abdominal surface is facing toward the ceiling.

Place the animal’s tail between the fourth and fifth fingers (ring and pinky) of the hand that is also holding the skin at the nape of the neck between thumb and forefinger.

Inject animal just proximal to the inguinal ligament with needle entering at an approximately 45° angle relative to abdominal surface. The needle should be pointed superiorly (see Note 13).

Repeat Steps 1–3 of the femoral artery ligation surgery.

The fur on the midline of the abdomen, from approximately the sternum to the level of the inguinal ligament, should be removed using clippers or depilatory.

Using the large, delicate serration forceps and small surgical scissors, a small incision (∼2–3 cm in length) through the skin only, should be made parallel to the aorta.

You will now see the ventral musculature of the intraperitoneal (IP) cavity. Again, make a small incision (∼2–3 cm in length) through the muscle wall (see Note 15).

Your field of view will now largely be encompassed by the intestinal tract. You will need to move aside the intestinal tract before visualizing the aorta and vena cava.

Have a small syringe (1–3 mL) filled with sterile saline available to moisten the surgical site as you are separating the aorta and vena cava from surrounding tissue.

Do not separate the aorta and vena cava from each other. Instead, using the small, angled, smooth platform forceps and/or the triangular surgical spears, separate the pair from the surrounding tissue (see Note 16).

A drop or two of saline should be used to provide lubrication during this procedure. When there is a clear tunnel beneath both the aorta and vena cava, take a length of 6-lb test fishing line (approximately double the length of your occlusion tube) and run it beneath the vessels until half is protruding from each side.

Thread both ends of the 6-lb test line through the occlusion tube and tie it off.

Secure the occlusion tube in one of the alligator clips on the soldering stand helper (Fig. 1c). The long portion of the occlusion tube should be pointing toward the head of the mouse (Fig. 3).

Gently manipulate the soldering stand arm such that the surface of the occlusion tube is ∼5 mm above the aorta and vena cava. Make sure not to put pressure on the vessels thereby blocking flow.

Using the 5-0 suture, suture the muscle and skin layer closed around the occlusion tube.

When the surgery is complete, elevate the anesthetic to 2–3% for ∼3 min in anticipation of transferring the animal to the imaging preparation area where a nose cone should be located with anesthetic already flowing via a carrier gas.

Transfer the animal to the imaging preparation area.

The rectal temperature probe should be inserted using lubricating jelly and taped in place.

Both the animal’s hindlimbs should be gently straightened and taped in this position (see Note 17).

The end of the occlusion tube outside of the RF coil should be supported so that it remains parallel to the body of the mouse thereby minimizing the possibility of the occlusion line becoming taut beneath the aorta and vena cava.

Tie the 14-lb test line (long enough to reach through the extent of the bore of the magnet) to the 6-lb test line running through the occlusion tube.

Place the animal inside the RF coil. If you have an RF coil with its active extent toward one end of the coil, only the animal’s hindlimbs up to the occlusion tube will be inside the RF coil (see Note 7).

Tape the 14-lb test line to a plastic dowel long enough so that it can be easily reached from the other end of the magnet. There should be a small extent of occlusion line between the end of the dowel and the end of the occlusion tube. You will need to make sure that this dowel is supported at both ends to keep it in line, i.e., at the same height, with the occlusion tube. This will minimize the possibility of tension being put on the 6-lb test occlusion line that is running beneath the aorta and vena cava (Fig. 3).