Feb 02, 2026
Stereotaxic Injection (SNc or STR)
- Per Svenningsen1,
- Daniel Dautan 1
- 1University of Southern Denmark
Protocol Citation: Per Svenningsen, Daniel Dautan 2026. Stereotaxic Injection (SNc or STR). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1nz5g5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 02, 2026
Last Modified: February 02, 2026
Protocol Integer ID: 242449
Keywords: ASAPCRN, Stereotaxic Surgery , Gene Therapy , AAV Injection , SNc, STR, precise stereotaxic injection procedure for delivery, precise stereotaxic injection procedure, stereotaxic injection, nanoliter injector, small craniotomy, synuclein, standardized postoperative care, skull, tissue damage, injection accuracy, stereotaxic frame
Funders Acknowledgements:
Collaboration Research Network
Aligning Science Across Parkinsons
Abstract
This protocol describes a precise stereotaxic injection procedure for delivery of viral vectors or α-synuclein preformed fibrils (αSyn-PFF) into the substantia nigra pars compacta (SNc) or striatum (STR) of adult mice (2–3 months old). Using a Kopf stereotaxic frame, Hamilton syringe, and nanoliter injector, animals are anesthetized with isoflurane, the skull is surgically exposed and carefully leveled, and target coordinates are identified relative to Bregma. Small craniotomies are drilled, and reagents are injected at defined depths and volumes using a slow-in/slow-out approach to minimize tissue damage and reflux. Wounds are closed under sterile conditions, followed by standardized postoperative care and recovery. Injection accuracy and spread are verified post hoc by histological analysis.
Materials
Reagents & Solutions
• AAV6-CAG-aSyn, Stock titer: 2.3 × 10¹¹ vp/µL;
• αSyn-PFF, Pre-sonicate according to your validated protocol (e.g., 30–60 s, amplitude 20%)
• Sterile saline (0.9%)
• Lidocaine/prilocaine cream for pre-incision analgesia (optional).
• Ophthalmic ointment for corneal protection.
• 70% ethanol for cleaning.
• Betadine for sterilization of surgical area.
• Sutures or wound clips
Instruments
• Kopf stereotaxic frame with mouse adaptor
• Isoflurane vaporizer + O₂ supply
• Heating pad
• Electric drill with micro-burr
• Hamilton syringe (NanoNeurons #7001)
• Nanoliter injector (WPI)
• Sterile drapes
• Scissors, forceps, scalpel
• Cotton swabs, sterile gauze
Troubleshooting
Problem
SNc-specific
Solution
• DV depth is critical:
o Too shallow → VTA contamination
o Too deep → cerebral peduncle / blood vessel damage
• Ensure skull leveling is perfect
• Use slow micropump speed to avoid tissue damage
Problem
Striatum-specific
Solution
• Craniotomy must be small and centered
• Inject at two depths to ensure rostro-caudal spread
• Avoid lateral ventricle breach (too medial or too dorsal)
Problem
General
Solution
• Always minimize air in the syringe → incorrect volume delivery
• Clean drill between surgeries to prevent cross-contamination
• Keep virus/PFF on ice but avoid >2 hour room-temperature exposure
Before start
Pre-Surgical Preparation
Reagents & Solutions
- AAV6-CAG-aSyn
Stock titer: 2.3 × 10¹¹ vp/µL
Aliquot into low-binding tubes keep on ice during surgery.
- αSyn-PFF
Pre-sonicate according to your validated protocol (e.g., 30–60 s, amplitude 20%).
Keep on wet ice; avoid freeze–thaw.
- Sterile saline (0.9%)
- Lidocaine/prilocaine cream for pre-incision analgesia (optional).
- Ophthalmic ointment for corneal protection.
- 70% ethanol for cleaning.
- Betadine for sterilization of surgical area.
- Sutures or wound clips
Preparation of the Mouse - Anesthesia
Place mouse in induction chamber:
3–4% isoflurane in O₂ for 1–2 min until deeply anesthetized.
Transfer to nose cone on stereotaxic frame:
Maintain at ~2% isoflurane
Confirm anesthesia depth (toe pinch)
Preparation of the Mouse - Positioning
Secure ear bars gently into external auditory canals
Verify the skull is stable and mouse is centered
Keep mouse on heating pad to maintain 37°C
Preparation of the Mouse - Eye & Analgesia
Apply ophthalmic ointment to both eyes
Reapply as needed throughout procedure to ensure sufficient lubrication.
Optionally apply local anesthetic cream to scalp (10 min prior)
Surgical Exposure of the Skull
Shaving
Shave entire top of head, approx 150x inscision sight
Clean area with 3× alternating betadine and ethanol
Incision
Make a ~1.5–2 cm midline incision using a scalpel
Clear connective tissue from bone using cotton swabs
Leveling the Skull
Using digital readout:
Position the manipulator tip on Bregma, set Z = 0
Move to Lambda, check DV difference
Adjust nose clamp until Bregma–Lambda are within ≤0.05 mm DV
This is essential for hitting SNc reliably.
Identification of Coordinates
Coordinates are relative to Bregma and dura surface:
SNc Injection Coordinates
AP: –3.6 mm
ML: ±1.5 mm
DV: –4.2 mm
Volume: 250 nL
Laterality:
Bilateral for behavioral/ephys
Unilateral for mapping
Striatum Injection Coordinates (two-depth injection for volume spread)
AP: +0.5 mm
ML: ±1 mm or ±2 mm
DV: –2.5 mm (first) and –3.5 mm (second)
Volume: 500 nL total per site
Drilling the Skull
Mark Injection Site
Using digital coordinates, lower the needle to hover above the skull
Mark the site using the needle tip or fine marker
Drill
Use micro-burr to create a small craniotomy (~0.5 mm)
Avoid damaging dura
Clean bone dust with sterile saline + cotton swab
Preparing the Hamilton Syringe
Load virus or αSyn-PFF slowly to avoid bubbles
Flick gently to remove air
Insert syringe into nanoinjector
Set injection rate corresponding to 10 min delivery:
Example: for 250 nL → 25 nL/min
For 500 nL → 50 nL/min
Injection Procedure
Lowering the Needle
Move needle to AP and ML
Lower slowly until touching dura
Zero the Z coordinate
Lower to target DV depth (SNc: –4.2 mm)
Injection (“slow-in slow-out” technique)
Begin nanoinjection: total duration 10 minutes
Maintain stable isoflurane (2%)
Do not move the animal or frame during injection
Post-Injection Diffusion Period
After injection completes:
Leave needle in place for 5 minutes to allow diffusion
Prevent backflow by maintaining stable positive pressure
Needle Withdrawal
Withdraw very slowly, over ~1–2 minutes
This dramatically reduces reflux, especially in striatum
Repeat for Bilateral Injections
Move to next coordinate and repeat drilling + injection
Maintain sterile field at all times
Closing the Wound
Rinse skull with sterile saline
Gently lower scalp over skull
Close with:
Sutures (preferred) or wound clips
Apply topical antibiotic ointment
Postoperative Care
Recovery
Move mouse to warmed recovery cage (37°C)
Maintain monitoring until animal is fully ambulatory
Do not leave unattended until righting reflex returns
Analgesia
According to IACUC approved protocol, adminisiter meloxicam and/or buprenorphine
Continue to give for 48-72 hours
Surgical Recovery Time
Allow at least 2 weeks before behavioral testing or physiology
Verification of Injection Accuracy - SNc / Str Target Validation
Perfuse at experimental endpoint
Perform TH immunostaining
SNc: dense TH+ dopaminergic zone
STR: characteristic TH+ terminal fields
Check for:
Injection tract position
Spread of virus/PFF
Hemispheric symmetry (if bilateral)