Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) Muscle
Transcrição
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.
