Prepare embedding molds by creating cylindrical tin foils for each sample. Fill the molds with optimum cutting temperature or OCT compound. Then prepare two insulation barrels.
Fill the small barrel with isopentane and the large barrel with liquid nitrogen. Place the small barrel inside the larger one 10 minutes before muscle dissection. Begin by preparing the mouse for dissection.
Cut open the skin on the face of the mouse to expose the masseter muscle. Remove the parotid glands to fully expose the posterior part of the masseter muscle. Dissect the masseter muscle from its anterior attachment up to its posterior attachment on the mandible.
Then submerge the muscle tissue in OCT, holding it perpendicular to the compound. Transfer the mold into the pre-chilled isopentane using a needle holder. Wait for 40 seconds until the OCT becomes white and solid.
Store the fresh frozen muscle samples in labeled 24-well plates. Section the samples immediately at 20 degrees Celsius, or store them at 80 degrees Celsius for long-term use. Next, cut open the skin from the ankle to the knee and remove the fascia to expose the tibialis anterior muscle.
Use the tip of the microdissection forceps to isolate the tendon of the tibialis anterior and slide it up to the knee to break off the fibers attaching to the tibia. Cut the tendon at the ankle. HE and sirius red staining revealed that upon freezing injury, the tibialis anterior muscle showed complete regeneration after 14 days with morphology similar to intact control.
In contrast, masseter muscle exhibited impaired myofiber regeneration and excessive extracellular matrix deposition after 14 days of freezing injury. Immunohistological staining for Pax7 after 14 days of injury showed densely populated nuclei but without proportionally increased muscle satellite cells. A large number of centrally located myofibers were detected in the tibialis anterior muscle at 14 days post-injury, while the contour of myofibers was barely noticeable in the masseter muscle in the injured area.
Instead, the infiltration of PDGFRalpha-positive FAPs was observed.
