Apr 01, 2026
Mouse stereotaxic intracranial injection
- Gerard Michael Coughlin1
- 1California Institute of Technology
Protocol Citation: Gerard Michael Coughlin 2026. Mouse stereotaxic intracranial injection. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbjor3vpk/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: March 30, 2026
Last Modified: April 01, 2026
Protocol Integer ID: 314133
Keywords: Mouse, Intracranial injection, Adeno-associated virus, AAV, Stereotactic surgery, mouse stereotaxic intracranial injection delivery of genetic cargo, stereotaxic intracranial injection delivery, specific brain regions through stereotaxic surgery, stereotaxic injection, basic guidelines for stereotaxic injection, stereotaxic surgery, mouse brain, specific brain region, fundamental technique in neuroscience, neuroscience, mouse, viral vector
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
Delivery of genetic cargo to specific brain regions through stereotaxic surgery is a fundamental technique in neuroscience. This protocol provides basic guidelines for stereotaxic injection of reagents (e.g. viral vectors) into the mouse brain.
Troubleshooting
Safety warnings
Isoflurane is a halogenated anesthetic gas associated with adverse health outcomes in humans and must be handled according to governmental and institutional regulations. To reduce the risk of occupational exposure during rodent anesthesia, waste gas was collected in a biosafety cabinet using a charcoal scavenging system as approved by the California Institute of Technology.
Ethics statement
Animal husbandry and all procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.
Before start
Ensure that you have the necessary approvals and training(s) before starting this protocol. Follow institutional and veterinary guidelines, and approved protocol(s) for your lab.
Mouse stereotaxic intracranial injection
Note on injection coordinates
Note
This protocol assumes that the injection will be performed using a needle perpendicular to the vertical axis. In same cases, it may be best to angle the needle to access a structure (e.g. if an unangled needle would pierce a ventricle). In these cases, depth measurement and craniotomy hole position(s) will need to be adjusted to account for angled injection.
Starting point injection coordinates for a target brain region can be determined using a stereotaxic atlas and/or consulting published literature. For particularly challenging targets (e.g. small regions, deep regions, etc.), it may be beneficial to practice injections using a dye such as Evans blue. Animals injected with the dye can be euthanized immediately, and the brain fixed and sectioned to determine targeting accuracy.
In some cases, it may be helpful to measure depth from the brain surface, rather than from bregma.
Note on surgical procedures
Note
Follow institutional and veterinary guidelines, and approved protocol(s) for your lab. This protocol is meant to give a general idea of how the surgery is done. We will not go into detail about particular anaesthesics used, as these are likely to vary between labs and institutions.
This surgery generally takes 1-2 hourse, depending on the number of injection sites. It is possible to perform the surgery on 2 animals simultaneously, provided you are set up to do so.
Ensure that you have the necessary approvals and training(s) before starting this protocol.
Anaesthesize the animal, provide analgesic(s) and other necessary medications according to approved protocol. Shave the animal's scalp, then transfer to a stereotaxic frame, with a heating pad to maintain body temperature. Once sufficient time has passed for anaesthesia and analgesia, fix the animal's head in the stereotaxic frame and level the head with the stereotax.
If applicable, apply local anaesthetics and/or eye ointments.
Clean the animals' scalp according to approved protocol.
Make an anterior to posterior midline incision to expose the animal's skull. Use sterile cotton swabs and sterile saline to clean the incision and skull, and to gently part the skin to reveal the animal's skull. Ensure that the skull is completely dry before proceeding.
Identify bregma and lambda on the skull. Using a drill bit attached to a drill mounted on a stereotax arm, level the head in the stereotaxic frame. It can be helpful to first level the skull by eye, then to use a drill bit and the stereotaxic frame to measure positions, as such:
- Check whether bregma and lambda have the same medio-lateral (ML) position, and if not, adjust their position using ear bars until they match
- Check whether bregma and lambda have the same dorso-ventral (DV) position, and if not, adjust their position until they match
- Identify two points 2.0 mm behind bregma, and 2.5 mm to either side of midline (i.e. AP -2.0 mm, ML ±2.5 mm). Check whether these points have the same DV position, and if not, adjust their position until they match
Unless the animals skull is perfectly mounted in the stereotax, adjustments in one dimension may affect other dimensions. Thus it may be necessary to repeat this process multiple times, making smaller and smaller adjustments each time.
Note
Many stereotaxic atlases include diagrams of bregma and lambda.
Once the animal's head is leveled, use a drill to make small craniotomy holes over the target region (i.e. use the same ML and AP coordinates as the injection site).
Note
Craniotomy holes placed directly over large surface blood vessels can produce lots of bleeding. In these cases, it may be best to use an angled injection strategy.
Fill a microinjection needle and microsyringe with the reagent to be injected. Mount the pump onto the stereotax arm and then attach the needle.
Note
For precise injection of small volumes, we use:
- NanoFil microinjection needles (33 or 35 Gauge blunt) and 10 μL Nanofil microsyringes (World Precision Instruments)
- Microsyringe pump (UMP3 from World Precision Instruments)
- Microsyringe pump controller (Micro4 from World Precision Instruments)
Using the controller, expel a small amount of the liquid. You should see a small droplet on the end of the needle. Remove this droplet using a sterile cotton swap.
Note
This step is to make sure that there is liquid in the microinjection needle, at the tip, rather than air.
Zero the needle tip on bregma or the brain surface (depending on what depth will be measured relative to), then lower the needle into the brain through the craniotomy hole(s) until the desired depth is reached.
Program the microsyringe pump controller to inject the desired amount of reagent, over a period of 5-10 min.
Using the micropump controller, inject the desired amount of reagent into the target region.
Note
*CRITICAL*
At the start of the injection, write down the approximate volume of liquid in the syringe. At the end of the injection, check that approximately the correct volume was expelled into the tissue.
Once the microsyringe controller has stopped injecting, wait at least 5-10 mins before moving the position of the microinjection needle.
Note
This allows the reagent to diffuse into the tissue, mitigating any potential backflow up the tract as the microinjection needle is withdrawn out of the tissue.
Move the microinjection needle to the next injection site and repeat the injection process. When withdrawing the microinjection needle, move the needle slowly to reduce backflow up the needle tract.
When moving between craniotomy holes, gently clean the microinjection needle with sterile saline and a cotton swab.
Once all the injections are done, close the incision with suture.
Transfer animal back to home cage, and provide any necessary post-operative medications and care.