To begin, place the vials of buprenorphine diluted in sterile, 0.9%saline, and notexin, diluted in PBS, on ice. Under the nose cone setup, shave the hind limbs of an anesthetized mouse with a trimmer, then transfer the mouse back into the induction box. Prepare insulin syringes with 15 microliters of notexin solution for GA, and 10 microliters of notexin solution for TA injections.
Once the mouse is moved to the nose cone set up, disinfect the injection site with an ethanol wipe. Insert the needle with the bevel facing downwards in the belly of the TA muscle at a 30 degree angle. Advance the needle parallel to the tibia to reach the mid-belly of the TA and inject the notexin solution slowly for 10 seconds.
To inject the notexin solution into the GA, insert the needle at a 45 degree angle in the mid-belly of the lateral head of the GA muscle and inject the solution. Turn the mouse around and, using an insulin syringe, inject buprenorphine subcutaneously. Calculate the volume of the iododeoxyuridine solution required for each mouse using this equation.
Using an insulin syringe, perform an intraperitoneal injection of iododeoxyuridine solution at the sterilized injection site 8 to 12 hours before euthanasia.
