TITLE: Stereotaxic Injections
AUTHORS: Lindsay Meyerdirk, Michael Henderson

[DESCRIPTION]
This protocol details stereotaxic injections for any purpose, but in this case is primarily used for the injection of misfolded proteins.

[GUIDELINES]
*All experiments involving animals must comply with federal and local law. All procedures should be reviewed and approved by the institutional animal care and use committee (IACUC).

[STEPS]
SECTION: Preparing Mouse
1. Turn on the heating box so the pad starts warming.
SECTION: Preparing Mouse
2. Make sure surgery area is sterilized then set up entire station, leaving all sterilized tools covered until ready to start procedure.
SECTION: Preparing Mouse
3. Anaesthetize mouse per IACUC protocol, mouse weight and genetic background.
SECTION: Preparing Mouse
4. Once mouse has reached an anesthetic plane, shave the head up to between the eyes. When using isoflurane place the mouse back into the chamber until it no longer responds to a toe pinch.
SECTION: Preparing Mouse
5. Place the mouse head in the stereotaxic mount by placing the teeth in the bite bar and sliding the cone over the nose. Once the mouse is not responsive to toe pinch, place the ear bars into the ear canal of the mouse very carefully so as to not crack the bone or puncture the tympanic membrane.
SECTION: Preparing Mouse
6. Cover the eyes generously with eye ointment.
SECTION: Preparing Mouse
7. Place a sterile drape over the entire mouse, and cut a small hole where the incision will be. 

SECTION: Preparing Mouse
9. Inject the 1:1 lidocaine/bupivacaine subcutaneous (SQ) at the injection site. Lift up the sterile drape and inject 250 µL of sterile saline SQ on each side of the mouse (500 µL total), and also inject buprenorphine SQ based on the mouses weight.  

SECTION: Preparing Mouse
10. Lift a small patch of skin at the base of the skull with forceps and cut rostrally in a vertical line from lambda to between the eyes.  

SECTION: Preparing Mouse
11. Clean skull with sterile cottons swabs.
SECTION: Preparing Mouse
12. Place needle in holder with the bevel facing toward the front. Needle holder should have an alignment of 0.
SECTION: Head Alignment
13. With needle directly over Bregma, write down anterior-posterior (A-P), left-right (L-R), dorsal-ventral (D-V) position.
SECTION: Head Alignment
14. Go to Lambda. Check the height here and level with Bregma to within 0.1 mm tolerance by raising or lowering the ear bars slowly and evenly.
SECTION: Head Alignment
15. Go 2 mm to the right of Bregma and record the D-V measure. Go 2 mm to the left of Bregma and record the D-V measure. Level the skull within a tolerance of 0.1 mm.
SECTION: Head Alignment
16. Move to the injection site. Make a V-shaped mark at the injection site.  

SECTION: Injection
17. Move the needle far from the injection site.
SECTION: Injection
18. Drill a hole with a 28 g burr (slightly bigger than the injection needle).
SECTION: Injection
19. Move the injection needle back into place. Carefully move the needle to the top of the dura, without puncturing the tissue.
SECTION: Injection
20. Calculate how far down to go.
SECTION: Injection
21. Lower needle to the site of injection.
SECTION: Injection
22. Inject material at 0.4 μL/min.
SECTION: Injection
23. Wait 3 minutes after the injection has finished
SECTION: Injection
24. Raise needle out of the brain.
SECTION: Injection
25. Use an iodine swab to moisten the edge of the incision site and tissue, careful not to get any iodine in the drilled hole. Suture the skin closed with 3-4 stitches. Each stitch has 3 knots. Front-to-back, back-to-front, front-to-back. Follow sutures with a small amount of VetBond to secure the wound together. 

SECTION: Post-operative Care
26. Place the mouse back into a clean cage on a warming pad and watch for the mouse to wake up.
SECTION: Post-operative Care
27. Once awake place a Tylenol water bottle (1 mg/mL) on the cage for the next 3 days.
SECTION: Post-operative Care
28. Monitor the mice once daily for open wounds or adverse side-effects, recording on the surgical card for 4320 min.

SECTION: Post-operative Care
29. After day 3 remove the water bottle.
SECTION: Preparing Mouse
8. Sterilize the Incision site by swabbing with 70% ethanol followed by Iodine three times.