Hindlimb Skeletal Muscle Extraction: A Procedure to Excise Hindlimb Skeletal Muscles from Murine Model

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tissue and Organ Excision

NOTE: For use of tissues in biochemical or molecular biology assays, plan to weigh each organ and tissue and place a fragment immediately into pre-labeled cryotubes. Snap freeze in liquid nitrogen and store at -80 °C.

1. In order to avoid contamination of tissue samples with fur, spray 70% ethanol over the entire body.
2. Place the mouse on a dissection bed in a supine position and extend a limb vertically by attaching the foot to an elevated buret clamp.
3. Grasp the skin of the lower limb with Dumont forceps, and use the curved fine scissors to gently remove the skin and fascia, exposing the underlying muscle bellies of the lower limb.
4. Identify the gastrocnemius muscle on the posterior lower limb by its origin at the lateral and medial condyles on the posterior femur and its insertion at the calcaneus via the calcaneal tendon.
   1. Proceed to cut the gastrocnemius calcaneal tendon. Grasp the distal end of the gastrocnemius with Dumont forceps and pull the muscle belly toward its origin. Using scissors, cut the muscle at the origin as close as possible to the femur. Place the muscle in a dish. Weigh it immediately and freeze it in a tube in liquid nitrogen.
      NOTE: Be sure not to excise the soleus muscle along with the gastrocnemius. If that occurs, carefully remove soleus by gently pulling it off with the forceps.
5. Proceed to identify and excise the tibialis anterior muscle from its origin at the anterolateral surface of the tibia and its insertion at the medial cuneiform via its distal tendon.
   1. In order to have leverage, hold the foot by grasping the digits with the index finger and thumb. Insert the tip of Dumont forceps immediately under the superficial distal tendon of the tibialis and move the forceps such that the blunt side can be used to detach the tibialis muscle belly from the underlying connective tissue.
   2. Cut the distal tendon using the fine curved scissors and then cut the muscle at the origin as close as possible to the tibia.

Materiais

Name	Company	Catalog Number	Comments
CD2F1 mice	Harlan	60
Anesthesia apparatus	EZ-Anesthesia	EZ-7000
OCT	Tissue-Tek	4583
Cryostat	Leica	CM1850
Cork disks	Electron Microscopy Sciences	63305
Microtainer	BD	365873
Superfrost plus glass slides	VWR	48311-703
Streptomycin-Penicillin	Cellgro	30-002-CI