TITLE: Stereotactic injection of viral vectors
AUTHORS: William S Conrad, Lucie Oriol

[DESCRIPTION]
Here we describe stereotactic injection of AAV into ventral tegmental area (VTA) of mice. 

[STEPS]
SECTION: A.   Pre-surgery Preparation
1. Pull pipettes and cut at 9.0 mm from start of glass bend with microdissection scissors.
SECTION: A.   Pre-surgery Preparation
2. Wash hands and put on clean lab coat and gloves
SECTION: A.   Pre-surgery Preparation
3. Turn on glass bead sterilizer, heating pads (for operation and recovery), stereotactic coordinate display, nanoject, and drill.
SECTION: A.   Pre-surgery Preparation
4. Wipe stereotactic frame and surrounding area with 70% EtOH.
SECTION: A.   Pre-surgery Preparation
5. Place autoclaved tools in sterile field.
SECTION: A.   Pre-surgery Preparation
6. Place autoclaved absorbent pad on top of operation heating pad and place in frame.
SECTION: B.	Animal placement and craniometry
7. Weigh mouse then place in induction chamber at 4% isoflurane for five minutes.
SECTION: B.	Animal placement and craniometry
8. Once mouse is anesthetized and displaying slowed, deep breathing, place mouse into nose cone of stereotactic frame with 2% isoflurane.
SECTION: B.	Animal placement and craniometry
9. Fix mouse into place with ear bars.
SECTION: B.	Animal placement and craniometry
10. Add ophthalmic ointment to eyes.
SECTION: B.	Animal placement and craniometry
11. Inject carprofen (5mg/kg s.c.)
SECTION: B.	Animal placement and craniometry
12. Wipe top of head with betadine.
SECTION: B.	Animal placement and craniometry
13. Check for anesthesia.
SECTION: B.	Animal placement and craniometry
14. Use scalpel to cut scalp and periosteum in one motion, in rostral to caudal direction.
SECTION: B.	Animal placement and craniometry
15. Gently push two sides of periosteum apart with autoclaved cotton swabs to expose skull.
SECTION: B.	Animal placement and craniometry
16. Clean skull with cotton swabs and sterile saline if needed.
SECTION: B.	Animal placement and craniometry
17. Once the skull is dry, attach drill and locate bregma using the stereotax binocular. Use the drill to lightly mark bregma. Zero the medial-lateral (ML), anterior-posterior (AP), and dorsal-ventral (DV) coordinates.
SECTION: B.	Animal placement and craniometry
18. Locate lambda. If change in AP axis is greater than 0.05 mm, adjust skull’s pitch. Return to bregma, re-zero coordinates, and locate lambda again.
SECTION: B.	Animal placement and craniometry
19. Ensure that there is less than 0.05mm of tilt in ML axis by measuring DV at AP: -2 between ML:+2 vs -2. Otherwise adjust skull’s tilt.
SECTION: B.	Animal placement and craniometry
20. Bring skull to ML and AP coordinates of region of interest. For VTA we used ML: ± 0.35 and AP: −3.4 mm. Turn drill on and slowly lower into skull. Stop before reaching dura.
SECTION: B.	Animal placement and craniometry
21. Wipe skull clean with sterile saline and cotton swabs.
SECTION: B.	Animal placement and craniometry
22. Pierce dura with the tip of a 32 gauge needle. Use eye-spears to stop bleeding. Clean area with saline and cotton swabs as needed.
SECTION: C. Viral Injection
23. Partially fill a 1 ml syringe with mineral oil, then plunge into pulled glass pipette until full. Ensure no bubbles are in pipette. Wipe clean with cotton swab. Be careful to not break pipette tip for the duration of the surgery. 
SECTION: C. Viral Injection
24. Insert glass pipet into nanoject and empty the nanoject
SECTION: C. Viral Injection
25. Remove drill from stereotactic holder and replace with nanoject. 
SECTION: C. Viral Injection
26. Pipet ~2 uL of virus onto parafilm.
SECTION: C. Viral Injection
27. Use nanoject controller to draw virus into pipette. Ensure no air is pulled into pipette. Mark oil/virus meniscus on glass pipette
SECTION: C. Viral Injection
28. Bring pipette to bregma and re-zero coordinates. 
SECTION: C. Viral Injection
29. Ensure pipette is working by injecting a small amount of virus with pipette tip visible. Wipe virus away with cotton swab.
SECTION: C. Viral Injection
30. Use coordinates to bring pipette to region of interest. Slowly lower pipette into brain. For VTA, we use coordinates ML: ± 0.35, AP: −3.4, DV: −4.4 mm relative to Bregma. 
SECTION: C. Viral Injection
31. Begin injection program. We inject 150 nl virus per side at 10 nl/sec, 5 sec delay between 15 injection cycles. 
SECTION: C. Viral Injection
32. Leave pipette in place for 10 minutes after injection. Slowly withdraw pipette by 0.05 mm, wait one minute, then slowly and complete withdraw pipette.
SECTION: C. Viral Injection
33. Try injecting a small amount of virus with the pipette tip visible to ensure no blood is clogging it. If no virus comes out, gently wipe tip with saline and cotton swab until the clog is removed. 
SECTION: C. Viral Injection
34. Ensure meniscus is lower than marked and mark it again if you continu with additional surgeries
SECTION: D. Suturing and recovery
35. Rehydrate skin near skull with saline.
SECTION: D. Suturing and recovery
36. Suture skin together.
SECTION: D. Suturing and recovery
37. Apply antibiotic ointment.
SECTION: D. Suturing and recovery
38. Remove animal from frame and place in recovery box with heating pad. Turn off isoflurane flow to stereotactic frame.
SECTION: D. Suturing and recovery
39. Once animal is ambulating and responsive, place it back into its home cage with clean bedding.
SECTION: D. Suturing and recovery
40. Monitor the animal’s weight and behavior for at least 5 days after surgery. Inject carprofen (5mg/kg s.c.) first day post-operation.
SECTION: D. Suturing and recovery
41. Wait at least 3 weeks for viral expression and surgery recovery.