Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells
Transcrição
On postoperative day 7, place the euthanized mouse on its ventral side and stabilize the distal limbs using pins. After removing the skin on the dorsal side of the injected limb and torso, use blunt dissection to expose the sciatic nerve deep to the muscles.
To access the nerve at the spinal cord region, separate the ilium and the sacrum by inserting closed scissors in the narrow area where the sciatic nerve is located, then open the scissors while holding the mouse. Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord.
Careful handling is essential as invaded nerves are extremely fragile and prone to breaking under tension or forceful handling. Be delicate and maintain the integrity of the sciatic nerve during the dissection.
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. Use a vernier caliper to estimate the gross length of invasion. Begin processing by embedding the dissected nerve in OCT compound. 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 and D. Then, place the embedded nerves on top of dry ice. Section samples using a Cryostat microtome at 5-micron thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve. After staining the slides with H&E and obtaining images with a slide scanner, use imaging software to quantify the length of invasion.
