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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Sciatic Nerve Extraction
- Take mice whose sciatic nerves are previously injected with cancer cells and euthanize them with CO2. Place the mouse on its ventral side and stabilize the distal limbs using pins.
- Remove the skin on the dorsal side of the injected limb and torso.
- Using blunt dissection, expose the sciatic nerve deep into the muscles.
- The nerve courses between the ilium and the sacrum. To have access to the nerve at the spinal cord region, separate the two bones. First, insert closed scissors in the narrow area where the sciatic nerve is located, and then open the scissors while holding the mouse. Be delicate and maintain the integrity of the sciatic nerve during the dissection.
- Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord.
NOTE: Invaded nerves are extremely fragile and prone to breaking under tension or forceful handling.
- Harvest the nerve by first cutting its distal end. Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end, as close as possible to its exit from the spinal column.
- Record then gross length of invasion using a Vernier caliper. This macroscopic estimation is only indicative.
2. Nerve Processing and Quantification
- Embed the dissected nerve in OCT compound. Make sure to place nerves longitudinally and as flat on the bottom of the mold as possible.
- Indicate on the cassette the proximal and distal side of the nerve by marking the letter P (Proximal) and D (Distal).
- Place embedded nerves on top of dry ice, if they are not sectioned immediately. Sample could be preserved at -80 °C for several weeks.
- Section samples using a Cryostat microtome at 5 μm thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve.
- Stain slides using H&E staining.
- Digitally scan stained nerve sections with a slide-scanner that provides high-resolution digital data.
- Using imaging software, quantify the length of invasion by clicking the measure distance button, area of invasion, or other desired parameters. For a good estimation of the length of invasion, use multiple sections (2 to 4) of the same nerve.