Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) Muscle
Transcrição
Spray all equipment and the hind limbs of the mouse with 70% ethanol. Use hardened fine-curved scissors and fine forceps to remove the skin and expose the underlying muscles. Remove the surrounding fascia with the fine-curved forceps without damaging the underlying muscles.
To remove the tibialis anterior or TA, grab the distal TA tendon with forceps, and cut it with fine scissors. While holding the TA at the tendon, pull it towards the knee and cut the muscle close to the knee to expose the EDL muscle.
Lift the distal EDL tendon with curved forceps, and cut with fine Vannas spring scissors. Expose the proximal EDL tendon by carefully pulling the EDL towards the knee. Then, cut the proximal tendon with scissors. Incubate the EDL muscles in the reaction tube at 37 degrees Celsius in a circulating water bath. Stop the digestion when muscles loosen up and single myofibers are visible.
Working under a stereo binocular microscope, equipped with a heating plate, flush the muscles with warm isolation medium, using the large bore pipette. Dissociate the muscles with the large bore pipette, until the desired number of myofibers are floating freely in the solution.
