To begin, with a pair of scissors and blunt forceps, lift and cut the skin of an anesthetized mouse over the right jugular vein. Cut a round window in the skin large enough to expose the jugular vein and some of the surrounding tissue. Gently push aside the adipose tissue, lymph nodes, and connective tissue using the blunt dissection technique.
Next, fill a 20 milliliter syringe with normal saline warmed to 37 degrees Celsius. Load the syringe into the syringe infusion pump. Then attach a 27 gauge butterfly needle and associated tubing to the syringe.
Set the syringe pump to a flow rate of 0.5 milliliters per minute to wash away blood from the punctured vein or artery. Position the stream of saline a few milliliters to the side of the puncture site and not directly over it. Once the jugular vein has been cleaned, start the timer.
Obtain 30 gauge and 33 gauge hypodermic needles for the jugular vein and the femoral artery respectively. Turn the bevel to face upwards. Then position the needle at a 25 degree angle over the jugular vein or femoral artery.
After noting the time, quickly puncture the top of the vessel. Note the time taken for the bleeding to stop completely. Wait for the formation of a thrombus before starting perfusion fixation.
Once perfusion has been completed, dissect the part of the jugular vein with the puncture site and thrombus. Use a pair of sharp scissors to make a diagonal cut across the distal end of the vessel segment and make a straight cut across the proximal end of the vessel segment. Next, place the fixed tissue at four degrees Celsius for 24 hours.
The next day, pour sodium cacodylate buffer solution into a 35 millimeter culture dish containing a five millimeter thick silicone mat. Carefully transfer the tissue into the buffer, ensuring that it is submerged in the buffer. With stainless steel minutien pins and a pair of fine forceps, carefully pin each end of the vessel to the mat.
Carefully clean the jugular vein. With a pair of micro scissors, cut the vessel wall opposite the puncture site lengthwise. Gently open the vessel, then pin it to the silicone mat with the intraluminal side facing up.
Remove the buffer solution from the dish. Then add 1%paraformaldehyde to submerge the vessel. Immediately place the lid on the dish to prevent drying out.
