Mar 26, 2025
Stereotactic Injections with Headframe Implant
- 1Allen Institute for Neural Dynamics
- Allen Institute for Neural Dynamics
Protocol Citation: Avalon Amaya, Katrina Nguyen 2025. Stereotactic Injections with Headframe Implant. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj72elx9/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: August 08, 2023
Last Modified: March 26, 2025
Protocol Integer ID: 86171
Keywords: Headframe, Head post, Rodent Neurosurgery, Stereotactic injections
Abstract
This protocol describes the surgical procedure, instrumentation, and reagents necessary for performing stereotactic injections and securing a headframe to an adult mouse brain for in-vivo recording procedures.
Image Attribution
Gabriel Rodriguez, Allen Institute.
Guidelines
Only perform this procedure in accordance with IACUC and veterinary requirements.
Materials
Anesthesia and other Drugs
IsofluranePatterson VeterinaryCatalog #07-890-8115
1 g DexamethasonebiorbytCatalog #orb134330
Ceftriaxone Injection MWI Animal HealthCatalog # 094311
Lactated Ringers Injection, USP, Preservative-Free, BaxterHenry Schein Animal HealthCatalog #059380
Ethiqa XR Buprenorphine Extended-Release Injectable Suspension for Mice and Rats 1.3mg/mL, 3mLFidelis PharmaceuticalsCatalog #099114
1 g AtropinebiorbytCatalog #orb322218
1 g CarprofenbiorbytCatalog #orb321211
Note
Drugs should only be administered in accordance with IACUC and veterinary requirements. Ensure timing, dosage, and route of administration are accounted for.
Surgical Tools and Supplies
| A | B | C | |
| Black handle scissors, ToughCut | Fine Science Tools | 14058-11 | |
| Scalpel handle | Fine Science Tools | 10003-12 | |
| Iris forceps | Fine Science Tools | 11064-07 | |
| Dumont #5 45° forceps | Fine Science Tools | 11251-35 | |
| 45° Vanna scissors, 8cm | World Precision Instruments | 500260 | |
| 45° or 90° Durotomy probe | Fine Science Tools | 10066-15 | |
| Plastic sterilization container | Fine Science Tools | 20810-02 | |
| Bulldog clamp | Fine Science Tools | 18053-28 | |
| PREempt Disinfectant spray | McKesson Corporation | 21101 | |
| 70% Ethanol (Diluted in-house) | Sigma Aldrich | 459836 | |
| Alchohol wipes | Becton, Dickinson and Company | 326895 | |
| Sterile Surgical Drape, 18x26 | Fisher Scientific | NC9517505 | |
| Sterile Multi-well plate, 24 well | Advantor | 29443-952 | |
| Nair hair removal cream | Arm & Hammer | 40002957 | |
| Betadine Solution 10% | McKesson Corporation | 1073829 | |
| Hemostatic Agent Surgifoam | McKesson Corporation | 403360 | |
| Sterile Gauze, 3x3” squares, (autoclave sterilized) | Patterson Veterinary | 07-893-8587 | |
| Cotton swabs, double ended, (autoclave sterilized) | Advantor | 89133-810 | |
| Sugi pointed sterile swabs | Fine Science Tools | 18105-01 | |
| Insulin syringes, U-100, 0.3 ml, 31G | Advantor | BD328438 | |
| Insulin syringes, U-100, 1 ml, 31G | Advantor | BD328418 | |
| Luer-Lock Syringe, 20 ml OR | Advantor | 53548-025 | |
| Luer-Lock Syringe, 10ml | Advantor | 75846-756 | |
| 25G 5/8-inch needle | Advantor | 89134-134 | |
| 32 mm Syringe Filter 0.2 µm Supor Membrane | Advantor | 75846-756 | |
| Press ‘n’ Seal | Medline | CLO70441 | |
| Saran Wrap | GLAD | Amazon B015CLAVU | |
| Sterile Drill Bits, 0.5/0.4, FG1/4 AND/OR | NeoBurr | 1734948 | |
| Sterile Drill Bits, 1.4/1.1, FG4 AND/OR | NeoBurr | 1734214 | |
| Sterile Drill Bits 1.0/4.2, EF4 | NeoBurr | 1730012 | |
| Sterile Scalpel blades, #10 OR | Advantor | 21909-378 | |
| Sterile Scalpel blades, #11 | Advantor | 21909-380 | |
| Systane Eye Ointment | Systane | Amazon ALCON293787 | |
| Artificial Cerebrospinal Fluid.V | Made in-house. Protocol referenced. | http://dx.doi.org/10.17504/protocols.io.besjjecn | |
| C Universal 4-META Catalyst, 0.7 ml | Parkell | S371 | |
| B Quick Base for MetaBond, 10 ml | Parkell | S398 | |
| Radiopaque L-Powder, white, 5 gm | Parkell | S396 | |
| Radiopaque L-Powder, clear 3 gm | Parkell | S399 | |
| Silicone implant coating, SORTA-Clear 18 | Renolds Advanced Materials | SORTA-Clear 18 | |
| Loctite 4305 | Henkel | 303389 | |
| XLite LED Curing light | Independent Dental | Flight Xlite2-CUR | |
| Vetbond Glue | Patterson Veterinary | 07-805-5031 | |
| Superglue, Singles | Krazy Glue | Amazon PK4 KG58248SN | |
| 3 ml transfer pipette, plastic | Avantor | 52947-970 | |
| Ortho-Jet BCA Liquid | Lang Dental Maufacturing Company | Ortho-Jet BCA Liquid | |
| Black cement (1) = 4 parts of Ortho-Jet BCA Powder (mixture) AND | Lang Dental Manufacturing Company | Ortho-Jet BCA Liquid | |
| Black cement (2) = 1 part of Powder tempura point, black | Jack Richeson & Co | 1# Black 62, Amazon B00JGZ8Q1A | |
| Kwik-Cast Sealant | World Precision Instruments | KWIK-CAST | |
| Heat-sterilized Glass pipettes AND/OR | Drummond Scientific | 3-000-203-G/X | |
| Heat-sterilized Glass pipettes | World Precision Instruments | 1B120F-4 | |
| “Marker” glass pipette, pulled, broken, and Sharpie mark for measuring coordinates | World Precision Instruments | 1B120F-4 | |
| Microcapillary Pipette tips | Eppendorf | 89009-310 | |
| Parafilm | Advantor | 52858-000 | |
| Lightweight Mineral Oil | Sigma-Aldrich | M8410 | |
| 30 gauge, 2" Backfilling Needle | Drummond Scientific | 3-000-027 | |
| Sterile Bone Wax | Central Infusion Alliance, Lukens | CIA2160287, 901 | |
| Sterilization pouches | Advantor | 89140-804 |
All tools / supplies can be substituted with their equivalent.
Key:
AND = Including the tool/supply in row below.
OR = Can use tool/supply in row below instead.
Autoclaved sterilized = Sterilized in-house.
mixture = Mix with tool/supply in row below.
Equipment
| Equipment | Manufacturer / Supplier | Part Number | |
| Small Animal Stereotaxic Instrument | Kopf | 1900 | |
| Adjustable Stage Platform | Kopf | 901 | |
| Stereo Microscope | Lecia | M80 | |
| Gooseneck Illumination | AM Scope | LED-6WA | |
| On-axis Illumination | Lecia | KL2500 LED | |
| Bead sterilizer | Sigma-Aldrich | Z378585 | |
| Small Animal Temperature Control System | CWE Inc. | TC-1000 | |
| Large Heat plate/pad | Lectro-Kennel | Outdoor Heated Pet Pad | |
| Dental Drill | NSK | Pana-Max2 M4 | |
| Oxygen Concentrator | Nidek Medical Products | Nuvo Lite Model 525 | |
| Isoflurane with oxygen delivery system | Patterson Scientific | Tec 3 EX | |
| Isoflurane induction chamber | Patterson Scientific | 78933385 | |
| Ear bars | Kopf | 1922 | |
| Ultra Fine Point Sharpie | Sharpie | 37001 | |
| Metabond ceramic mixing dish | Parkell | S387 | |
| Electrode Holder | Kopf | 1970 | |
| Galaxy Mini Centrifuge | Avantor | 76269-066 | |
| P20 Pipettor | Gilson | F123600 | |
| Silver wire | Stoelting | 50880 | |
| Midgard Precision Current Source | Stoelting | 51595 | |
| Nanoject II Variable Volume (2.3 to 69 nL) Automatic Injector OR | Drummond Scientific | 3-000-204 | |
| Nanoject III Programmable Nanoliter Injector | Drummond Scientific | 3-000-207 |
All equipment can be substituted with their equivalent.
Key:
OR = Can use equipment in row below instead.
Materials/Equipment designed/made in-house (CAD available upon request):
| Material | Part Number | |
| Titanium 42 Headpost * | 0160-100-42 | |
| Titanium AI Straight Bar * | 1365-6428-001 | |
| Titanium Headpost * | 0160-100-10 | |
| Titanium LC / Brainstem Headpost * | 0160-100-52 | |
| Dual Hemisphere Headframe * | 0160-100-57 | |
| Bregma Stylus | 0251-900-04 | |
| Lambda Stylus | 0111-300-01 | |
| Dovetail Clamp | 0111-200-00 | |
| Ear bar Headframe Clamp | 0155-100-00 |
All equipment can be substituted with their equivalent.
* = Optional
Personal Protective Equipment (PPE):
| Suggested PPE | |
| Gloves | |
| Disposable lab coat | |
| Disposable face mask | |
| Shoe covers / surgery shoes | |
| Scrubs | |
| Surgical cap | |
| Biohazard sharps disposal container | |
| Biohazard waste disposal container | |
| Blue light blocking glasses |
Utilize PPE in accordance with IACUC and veterinary requirements. Ensure sterility when necessary.
Safety warnings
- Personal Protective Equipment (PPE) should be used at all times while operating this protocol.
- Isoflurane Warning: Acute over-exposure to waste anesthetic gases (WAG) may cause eye irritation, headache, nausea, drowsiness or dizziness. Repeated exposure may cause damage to cardiovascular system and central nervous system. Refer to MSDS for additional information. Consult the surgical workstation guide to ensure all parts of the dispensation rig are functioning properly.
- Blue-light filter safety goggles must be worn while using LED curing light.
Ethics statement
Research focused rodent neurosurgery must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s).
This protocol has been approved by the Allen Institute Animal Care and Use Committee (IACUC).
PHS Assurance : D16-00781
AAALAC : Unit 1854
Before start
Reference protocol below for all general setup and takedown procedures for rodent neurosurgery:
Protocol
NAME
General Setup and Takedown Procedures for Rodent NeurosurgeryCREATED BY
Ali Williford
Reference protocol(s) below if performing injections:
Stereotaxic Surgery for Delivery of Tracers by Iontophoresis V.6: dx.doi.org/10.17504/protocols.io.14egn8ewzg5d/v6
Stereotaxic Injection by Nanoject Protocol V.6: dx.doi.org/10.17504/protocols.io.bp2l6nr7kgqe/v6
Expose and Prepare the Skull Surface
Expose and Prepare the Skull Surface
10m
10m
After hair removal and disinfection, create a midline incision with a scalpel blade from approximately behind the eyes to the front of the ears.
Illustration of initial skin incision on mouse head.
Mouse fixed to stereotaxic instrument base via bite bar and ear bars. Fur from top of head has been removed and skin has been sterilized (see General Setup and Takedown Procedures).
Using Vanna scissors, cut a teardrop shape of skin away, ensuring enough skull is exposed for headframe placement.
Illustration of skin removal on mouse head.
On the anterior side expose the rostral rhinal vein between the eyes.
On the left and right lateral sides stop at the interface between the skull and cheek muscle.
Posteriorly expose about 1 mm - 2 mm of neck muscle.
Remove exposed periosteum by rubbing it apart with cotton swabs and bunching it near the edges of the skin. Then cut away with vanna scissors at the skin edge. Use ACSF to rehydrate if necessary (i.e. periosteum dries out).
Note
Utilize a 10mL or 20mL syringe, 25G 5/8-inch needle, and a syringe filter to store and dispense ACSF.
Detach visible neck muscles using dumont forceps.
Score entire skull with scalpel blade to provide greater surface area for bonding agents. Do not obstruct any landmarks.
Seal all along incision site with Vetbond. Use a Sugi Absorption Spear to absorb any excess fluid either prior or during. Extend vetbond seal 1-2mm past incision site along undamaged skin.
Note
Extending the vetbond seal over healthy tissue delays the formation of exudate. Additionally, unsealed soft tissue will weep fluid that can compromise the attachment of the headframe.
Align The Skull
Align The Skull
5m
5m
Locate Bregma and Lambda landmarks with Dovetail Clamp and Bregma Stylus, and use them to level the skull in the anterior-posterior axis within 0.1mm.
Illustration of Bregma and Lambda landmarks.
If Lambda-Bregma offset is greater than 0.1mm in X, use the yaw adjustment on the stereotaxic alignment system to adjust the yaw within 0.1mm.
At midline, approximately midway between Bregma and Lambda, measure 2mm laterally on both the left and right hemisphere, ensuring that the skull is level in the medial-lateral axis within 0.15mm.
Perform Stereotactic Injections
Perform Stereotactic Injections
10m
10m
Skip to section "Headframe Placement" if not performing virus injections.
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject:
Begin at step 8.2.5"Mark the Injection Site"
Stop at step 8.5 "Suturing"
Reference protocol Stereotaxic Surgery for Delivery of Tracers by Iontophoresis V.6 for performing stereotactic injections via Iontophoresis:
Begin at step 8.2.6 "Mark the Injection Site"
Stop at step 8.5 "Suturing"
If target coordinates require an angled injection, rotate mouse before proceeding with the injection. Return to level once injection has been completed and pipette has been removed.
Fill burr holes with bone wax or Kwick-Cast.
Note
Ensure wax or Kwick-Cast is only within burr hole. Residue from wax or silicone on skull can compromise Metabond seal.
Headframe Placement
Headframe Placement
20m
20m
Seal entire skull with Vetbond.
If desired, mark coordinates on skull with Sharpie.
Note
It is often useful to mark coordinates indicating either Bregma, Lambda or future location of burr hole craniotomy.
Attach Dovetail Clamp to stereotaxic arm and attach stylus. Center stylus over headframe fiducial point and then raise stylus slightly.
Note
Depending on the headframe or head bar desired, the fiducial point may vary. For example the Titanium LC/Brainstem headframe utilizes the Bregma stylus with Bregma as a fiducial point.
Replace the stylus with the headframe and lower until there is contact with the skull.
Image of DHC headframe lowered onto skull.
Apply white Metabond around the outside of the headframe. Metabond should wick under and into the headframe. If it becomes too viscous, remake it. Place Metabond so the majority of the skull within the well remains exposed. Let dry completely (around 00:05:00 ).
10m
Use clear Metabond to seal the rest of the skull within the inside well. Allow all metabond to completely dry (around 00:05:00 ).
Image of completed surgery with well.
5m
Remove mouse from stereotaxic instrument base once metabond is fully dry.
Detach Dovetail Clamp from headframe carefully.
Remove earbars.
Remove mouse teeth from bitebar by lightly scruffing mouse in a way that raises the teeth out of the bite bar.
Recover mouse and takedown
Recover mouse and takedown
Obtain the mouse’s postoperative weight.
Place the mouse back in a recovery cage and put the cage on the 37 °C heat plate.
Reference General Setup and Takedown Procedures for Rodent Neurosurgery protocol for takedown procedure.