Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles
Transcrição
Place the ventral brain side facing up in a chamber containing cold dissection solution to isolate parenchymal arterioles. To isolate parenchymal arterials, secure the isolated brain with steel pins and cold dissection solution in a Petri dish containing more than 50-centimeter deep charcoal-infused silicone polymer coating.
Cut a rectangle of brain tissue from both hemispheres with sharp and aligned dissection scissors around the MCA, while ensuring that the upper part of the tissue segment is past the branching point from the circle of Willis. Secure the brain tissue into the dish with the MCA facing upwards with steel pins. Carefully make a shallow incision near the pins, and remove the pia with small forceps, gently peeling from one end towards the other.
Carefully secure the isolated pia with parenchymal arterioles branched from MCA in the dish with the pins, and carefully dissect the parenchymal arterioles. Cut off any remaining distal branches and ensure the arteriole is clean with no tissue attached. Use this clean intact arterial for enzymatic digestion. Alternatively, cut each arterial into two pieces for enzymatic digestion to prepare endothelial tubes, if desired.
For partial digestion of arteriolar segments, place intact arteriolar segments into 1 milliliter of dissociation solution in a 10-milliliter glass tube containing papain, dithioerythritol, collagenase, and elastase. Incubate arteriolar segments at 34 degrees Celsius for 10 to 12 minutes.
To isolate the arteriolar endothelial tube, place the trituration pipette attached with the microsyringe injector in the dissociation solution in the chamber and position it close to one end of the digested vessel segment. Set a rate within the range of 1 to 3 nanoliters per second on the pump controller for gentle treat duration. While maintaining 100 times to 200 times magnification, withdraw the arterial segment into the pipette, and then inject to dissociate the adventitia and smooth muscle cells. Triturate the vessel segment until all smooth muscle cells are dissociated and only endothelial cells remain as an intact tube.
