May 21, 2026
Co-injection of α-synuclein PFFs and retrovirus for retro labeling in mouse striatum
- Dayana Pérez-Acuña1,
- Thomas Biederer1
- 1Yale University
Protocol Citation: Dayana Pérez-Acuña, Thomas Biederer 2026. Co-injection of α-synuclein PFFs and retrovirus for retro labeling in mouse striatum. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5yq2dl1b/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: April 17, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 315277
Keywords: ASAPCRN, synuclein pff, synuclein, retrovirus for retro labeling, mouse striatum this protocol, fibrils with retro gfp, aav into the striatum, retrovirus, mouse striatum, retro labeling, retro gfp, striatum
Funders Acknowledgements:
ASAP
Grant ID: 020616
Abstract
This protocol describes the procedure to co-inject alpha-synuclein preformed fibrils with retro GFP-AAV into the striatum for tracing of pathology.
Materials
- Sonicated mouse α-synuclein pre-formed fibrils (PFFs)
- pAAV-CAG-GFP was a gift from Edward Boyden (Addgene viral prep # 37825-AAVrg ; http://n2t.net/addgene:37825 ; RRID:Addgene_37825)
- Sterile PBS appropriate for in vivo injection
- Isoflurane
- Isoflurane vaporizer
- Sterotaxic frame
- drill
- Eye lubricant (Optixcare Aventix)
- Povidone
- Ethiqa XR
- Hair trimmer
- Depilatory cream
- ethanol
- Drugs
- Injector pump (Harvard Apparatus; Pump 11 Elite)
- 10ul Hamilton syringe (Hamilton 1701RN),
- medical grade (Tygon AAD04091) tube
- 30G needle.
- Absorbable suture
Preparation of Injection Material
Dilute recombinant mouse α-synuclein PFFs to 2 mg/ml with sterile PBS
Note
Only use high quality PBS suitable for in vivo work.
Preparation of Injection Material
Sonicate PFFs as indicated in the protocol (https://dx.doi.org/10.17504/protocols.io.14egn69qql5d/v1)
Preparation of Injection Material
Add AAV Retrograde virus, AAVrg/GFP, to sonicated PFFs to a final titer of 8x108 GC/μl
Striatal Injection
Anesthetize mouse with 2-3% isoflurane with an isoflurane vaporizer
Once anesthetized (i.e., unresponsive to pain and with normal breathing rate), transfer mouse
to the stereotaxic frame fitted with a heating pad set to 37°C
Note
Position the nose cone for isoflurane delivery correctly to maintain the level of anesthesia
Apply eye lubricant to both eyes to prevent corneal damage
Shave fur above the skull with a hair trimmer.
Remove remaining fur by applying depilatory cream for one minute.
Note
Depilatory cream can burn the skin if applied longer than 1 minute.
Remove the cream and clean the area with 70% ethanol.
Clean the area with povidone.
Pain Management:
Inject 30 μl of local anesthetic (lidocaine, bupivacaine) subcutaneously along the incision site.
Administer preoperative Ethiqa XR 3.25 mg/kg.
Make an incision in the skin above the skull, and clean the area with a dry cotton swab.
Locate the dorsal striatal injection site relative to Bregma: AP +1.0 mm, ML +1.5 mm, DV
+3.0 mm from the dura mater.
Drill a hole at the desired coordinates without causing damage to the brain.
Be careful not to break the dura to prevent excessive bleeding.
Inject 2.5 μl of the PFF with AAVrg/GFP mixture (5 μg of PFF and 2× 109 GC total), with a rate of 0.35 μl/min with an injector pump.
Note
Our injection setup is a Pump 11 Elite (Harvard Apparatus) with a 10 μl Hamilton syringe (Hamilton, 1701RN) connected to a medical-grade tubing (Tygon, AAD04091) equipped with a 30G needle.
Wait 5 minutes after the full volume is injected. Then slowly withdraw the needle injection before removing the needle.
Close the incision with absorbable sutures, and administer Carprofen (5 mg/kg,
Subcutaneous)
Acknowledgements
pAAV-CAG-GFP was a gift from Edward Boyden (Addgene viral prep # 37825-AAVrg ; http://n2t.net/addgene:37825 ; RRID:Addgene_37825)