After sacrificing the mouse, place it on a foam surface and secure the forelimbs with tape or pins. Use operating scissors to cut both hind limbs over the knees. Transfer each hind limb to a separate dissection chamber, and add cold Tyrode's solution to cover the tissue.
Pin the first hind limb to the dissection chamber, ensuring the posterior face of the legs is visible. Under magnification, remove the skin, then expose and excise the flexor digitorum brevis or FDB muscle. Store the FDB muscle in a labeled glass vial containing one milliliter of Tyrode's solution.
Using fine scissors, separate the gastrocnemius muscle, then excise the soleus muscle. Remove the skin from the anterior face of the leg. Then, identify the distal tendons of the tibialis anterior and the extensor digitorum longus or EDL muscles in the ankle region.
Remove and discard the tibialis. Then, cut the distal tendons of the EDL and continue the dissection to completely remove the EDL muscle. After identifying the extensor hallucis longus or EHL muscle, begin the dissection following the tendon to the first digit.
Next, identify and follow the most external tendon of the peronei to sever it and remove the peroneus longus or PL muscle. Identify and trace the tendon to the fourth digit. Then, cut and remove the peroneus digiti quarti or PDQA muscle.
After dissecting the second hind limb, collect muscles of the same type in a labeled glass vial containing Tyrode's solution. Replace the Tyrode's solution in the dissection chamber to remove debris and mouse fur. Pour the PL muscles into the dissection chamber and verify their integrity and contraction.
Perform longitudinal or diagonal cuts on the soleus muscle following the central tendon, cutting approximately 80%of its length. For the EDL muscle, follow one or two tendons and cut about the same length as the soleus. Transfer each pair of muscles to a new glass vial containing three milligrams of collagenase type 2 in dissociation solution, and incubate them in a 37 degree Celsius water bath for 65 to 90 minutes with gentle shaking.
After 65 minutes of incubation, check the vial under stereoscope every five minutes. Once the muscle appears rippled and loose, and fibers begin to detach, rinse the muscles with Tyrode's at room temperature to deactivate and remove the collagenase. Using a set of fire-polished Pasteur pipettes, agitate the solution around the muscle with a five millimeter tip pipette.
Then, gently pull the muscles in and out of the tip three to four times. As the muscles starts to release fibers and becomes thinner, using a four millimeter tip, repeat the procedure to separate more fibers.
