This article outlines a method for isolating myofibers from mouse hindlimb muscles, specifically the tibialis anterior and extensor digitorum longus. The process involves surgical removal of muscle tissue, enzymatic digestion, and careful dissociation of myofibers for downstream analysis.
Begin with a euthanized mouse in the supine position. Sterilize its hindlimb. Surgically remove the skin and fascia—a thin casing of connective tissue beneath the skin—to expose the underlying hindlimb muscles.
Locate the tibialis anterior or TA muscle on the lateral side of the tibia. Holding the TA distal tendon, detach the muscle from the underlying tissue. Cut the muscle close to the knee to excise the TA muscle, revealing the underlying extensor digitorum longus or EDL muscle located in the anterior compartment of the hindlimb.
Incise the distal EDL tendon and pull the EDL muscle towards the knee. Cut the proximal EDL tendon to excise the EDL muscle. Transfer the harvested muscle to a tube with prewarmed media containing collagenase enzymes and incubate for the appropriate duration. Collagenases break down the connective tissue surrounding each muscle fiber or myofiber within the muscle. This digestion step loosens the myofibres from the inner layer of the muscle.
Following incubation, carefully transfer the digested muscle to a multi-well plate containing prewarmed media to increase the survival rate of myofibres. Transfer the plate to the heated stage of a stereo microscope. Now, gently triturate the digested muscle to dissociate and release the individual myofibers into the solution.
The isolated EDL myofibres can be used for further downstream analysis.
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.
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