TITLE: Mouse stereotaxic surgery
AUTHORS: madalynn.erb

[DESCRIPTION]
This protocol details the mouse stereotaxic surgery.

[STEPS]
SECTION: Procedure
1. Turn on the heating pad to and set to 36.9 °C.
SECTION: Procedure
2. Weigh mouse and record weight before starting surgery.
SECTION: Procedure
3. Place mouse in isoflurane chamber (3% isoflurane).
SECTION: Procedure
5. Place mouse back into isoflurane chamber (3% isoflurane) until fully anesthetized.
SECTION: Procedure
6. Transfer mouse onto stereotaxic frame and place the mouth / nose into nose cone with isoflurane.
SECTION: Procedure
7. Apply sterile ophthalmic ointment to eyes using a sterile swab (this will prevent desiccation).
SECTION: Procedure
4. After mouse is fully anesthetized, use hair clippers to shave head.
SECTION: Procedure
3.1. Record time of anesthesia.
SECTION: Procedure
6.1. Turn down isoflurane to 2%.
SECTION: Procedure
8. Use ear bars to secure mouse in stereotaxic frame ensuring head is level in all directions.
SECTION: Procedure
9. Using a sterile swab apply 70% EtOH to the scalp.
SECTION: Procedure
10. Using a sterile swab, apply povidone-iodine solution to the scalp.
SECTION: Procedure
10.1. Wait for povidone-iodine solution to dry before making surgical incision.
SECTION: Procedure
11. Perform a subcutaneous injection of Ethiqa XR (buprenorphine extended-release) into the leg.
SECTION: Procedure
11.1. Use 0.05 mL per 20 g mouse.
SECTION: Procedure
12. Inject 30 µL of Marcaine in 2-3 locations underneath the scalp near the incision site. This is a local analgesic.
SECTION: Procedure
12.1. Wait 30 seconds  - 1 min for the Marcaine to diffuse before performing incision.
SECTION: Procedure
13. Using a sterile scalpel, make a surgical incision to expose the skull.
SECTION: Procedure
13.1. Minimize the size of the incision as much as possible.
 

SECTION: Procedure
14. Position your injection needle at bregma and save location in the AP and XY axis.
SECTION: Procedure
15. Enter and save injection coordinates into the motorized stereotaxic frame.
SECTION: Procedure
15.1. Coordinates for right substantia nigra
Anterior-posterior (AP):  
Medial-lateral (ML):  
Dorso-ventral (DV): -4.2
SECTION: Procedure
16. Raise the needle away from the skull slightly and move to AP and ML injection coordinates.
SECTION: Procedure
17. Slowly lower the needle to touch the skull. Raise the needle and drill a small hole where the needle touched the skull.
SECTION: Procedure
18. Once the hole is drilled lower the needle to the surface of the brain ensuring that the needle is not deflected by the skull.
 

SECTION: Procedure
17.1. Slowly drill through the skull keeping the drill shallow enough to not damage brain tissue.
SECTION: Procedure
19. Raise the needle   -   providing space to flush the needle and load with virus.

SECTION: Procedure
19.1. Flush the needle with sterile Room temperature H2O.
Cover the mouse’s head with sterile gauze to absorb the H2O.
SECTION: Procedure
19.2. Draw up a 1 µL air bubble.
SECTION: Procedure
19.3. Load needle with 2.5 µL of virus.
Slowly draw up virus and watch the air / liquid interface to determine volume.
Needle / tubing should be marked with 1 µL intervals using a sharpie to help with this step.
SECTION: Procedure
20. Move the prepared needle down to the surface of the brain. Move the needle to the desired DV coordinate at a slow speed (250µm / sec).
SECTION: Procedure
21. Wait 30 s and then start the virus injections at 0.2 µL / min.
SECTION: Procedure
22. After the injection is finished wait for 5 min, leaving the needle in place and allowing the virus to diffuse away from injection site.
SECTION: Procedure
23. Remove the needle at slow speed (250 µm / sec).
SECTION: Procedure
24. Move the needle up away from the skull and then release it and rotate it out of the way.
SECTION: Procedure
25. Close the incision site using sterile forceps and surgical glue.
SECTION: Procedure
26. Administer 0.5 mL sterile saline solution subcutaneously to avoid dehydration.
SECTION: Procedure
27. Place the mouse back into the home cage and monitor recovery.
SECTION: Procedure
27.1. Record time at recovery.
SECTION: Procedure
28. Monitor post operation recover for 4320 min and record any observations of pain or distress onto surgery cards.