May 13, 2026
Motor Cortex Cranial Windowing
- Conor Grasso1,
- Benjamin Ouellette2,
- Avalon Amaya2,
- Robyn Naidoo2
- 1Allen Institute for Neural Dymanics;
- 2Allen Institute for Neural Dynamics
- Allen Institute for Neural Dynamics
Protocol Citation: Conor Grasso, Benjamin Ouellette, Avalon Amaya, Robyn Naidoo 2026. Motor Cortex Cranial Windowing. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxdbrgx1/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: January 23, 2024
Last Modified: May 13, 2026
Protocol Integer ID: 94026
Keywords: M1, Motor Cortex, Craniotomy, Mouse, Durotomy, Rodent Neurosurgery, Headframe, Precision laser leveling, Headpost, injections within the cranial window, motor cortex cranial windowing this protocol, cranial windowing surgery, cranial window over the motor cortex, motor cortex cranial windowing, cranial window, craniotomy, stereotactic injection, craniotomy coverslip, surgical procedure, mouse motor cortex, stabilizing headframe, variations of stereotactic injection, cortical damage, timing of injection, procedure, motor cortex, headframe, precision laser leveling, skull, injection, neural imaging
Abstract
This protocol describes the surgical procedure, instrumentation, and reagents necessary for the installation of a stabilizing headframe and cranial window over the motor cortex of an adult mouse. This protocol should be utilized for neural imaging and experimentation regarding the mouse motor cortex.
This procedure accommodates for 2 variations of stereotactic injections (Optional 1 and Optional 2). Specifically this protocol accommodates for injections before and after the craniotomy and durotomy. Timing of injections during cranial windowing surgeries is typically dependent on their coordinates. Injections outside of the cranial window should be performed prior to the craniotomy and durotomy to allow for adequate burr hole sealing. Injections within the cranial window should be performed after the craniotomy and durotomy to minimize cortical damage.
Additionally, this protocol utilizes precision laser leveling, which involves a non-standard operation order where the headframe is leveled based off the craniotomy coverslip rather than the skull.
Image Attribution
Gabriel Rodriguez, Allen Institute
Guidelines
Only perform this procedure in accordance with IACUC and veterinary requirements.
This protocol has been written for review purposes only, and should not be utilized for training. Users of this protocol should be familiar with common vocabulary and technique for rodent neurosurgery.
Materials
Anesthesia and other Drugs
IsofluranePatterson VeterinaryCatalog ##07-890-8115
1 g DexamethasonebiorbytCatalog #orb134330
Ceftriaxone InjectionAmerisourceBergen MWI Animal HealthCatalog #094311
Lactated Ringers Injection, USP, Preservative-Free, BaxterHenry Schein Animal HealthCatalog #059380
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:
| Tool / Supply | Manufacturer / Supplier | Part Number | |
| 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 | |
| large iris forceps | Fisher Scientific | 13-820-073 | |
| 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 | Avantor | 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) | Avantor | 89133-810 | |
| Sugi Absorption Spear | Fine Science Tools | 18105-01 | |
| Insulin syringes, U-100, 0.3 ml, 31G | Avantor | BD328438 | |
| Insulin syringes, U-100, 1 ml, 31G | Avantor | BD328418 | |
| Luer-Lock Syringe, 20 ml OR | Avantor | 53548-025 | |
| Luer-Lock Syringe, 10ml | Avantor | 75846-756 | |
| 25G 5/8-inch needle | Avantor | 89134-134 | |
| 32 mm Syringe Filter 0.2 µm Supor Membrane | Avantor | 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 | Avantor | 21909-378 | |
| Sterile Scalpel blades, #11 | Avantor | 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 | |
| CS-5R Coverslips, 5 mm | Warner Instruments | 64-0700 | |
| 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-Sil Sealant | World Precision Instruments | KWIK-SIL | |
| 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 | Avantor | 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 | Avantor | 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 | Leica | 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 | |
| 3mm Cranial Window (two 3mm stacked with single 4mm circular cover glass lip) | Tower Optical 18687-2 | |
| Mesoscope Well | 0160-200-20 | |
| Neuropixel Well | 0160-200-45 | |
| Well Cap | 0160-055-09 | |
| Bregma Stylus | 0251-900-04 | |
| Dovetail Clamp | 0111-200-00 | |
| -0⁰ Ear bar Headframe Clamp | 0155-110-00 | |
| Prober Holder | 0155-200-00 | |
| Titanium MotorCtx Headpost | 0160-100-54 | |
| Laser Leveling tool | 0111-500-00 |
All equipment can be substituted with their equivalent.
Personal Protective Procedures (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 |
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
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:
Optional 1:
For stereotactic injections prior to performing the craniotomy and durotomy, follow section titled with "Optional 1" in addition to the standard sections.
Optional 2, 2.1, 2.2:
For stereotactic injections after performing the craniotomy and durotomy, follow sections titled with "Optional 2", "Optional 2.1" and "Optional 2.2" in addition to the standard sections.
This protocol utilizes precision laser leveling, which involves a non-standard operation order where the headframe is leveled based off the craniotomy coverslip rather than the skull.
Graphical Overview of Procedure
Map diagram of protocol workflow.
Expose and Prepare Skull Surface
10m
After hair removal and disinfection, create 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 stereotactic 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.
Illustration of skin removal on mouse head.
On the left side, remove enough skin to expose 1-2mm of cheek muscle. On the right side, do not expose the cheek muscle. Anteriorly, stop 1-2mm anterior of the rostral rhinal vein.
Remove exposed periosteum by rubbing apart with cotton swabs and bunching near the edges of the skin. Then cut away with Vanna scissors at the skin edge. Use Artificial Cerebrospinal Fluid (ACSF) to rehydrate if necessary (i.e. periosteum dries out).
Note
Utilize a 10mL or 20mL syringe, 25G 5/8-inch needle, and a 0.2um syringe filter to store and dispense ACSF.
Detach 3mm of cheek muscle just posterior of left eye with tip of Dumont forceps. Do not remove more than 1mm deep into tissue. Be mindful not to damage the muscle structure of the eye.
Cropped photo of mouse skull displaying extent of muscle removal.
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 healthy 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
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 stereotactic alignment system to adjust the yaw to within 0.1 mm.
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.
Optional (1): Perform Stereotactic Injections Outside of the Cranial Window
20m
Optional 1
This optional section accommodates injections prior to performing the craniotomy and durotomy. Typically, this is done for injections that will be outside the craniotomy window, as placement of the headframe will obstruct most coordinates. Injection coordinates within the craniotomy window are performed after the craniotomy and durotomy as it will minimize cortical damage.
Skip this section if you will not be performing injections prior to the craniotomy and durotomy. .
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject:
Begin at step 8.2.6 "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".
Once completed with the injection, fill burr hole with bone wax with the broken end of a cotton swab.
Note
Do not leave excess wax residue on the skull. Excess wax can contribute to an insufficient seal between the skull and the headframe.
Optional (2): Mark Stereotactic Injection Coordinates for After Craniotomy and Durotomy.
Optional 2
This optional section accommodates injections that will be performed after the craniotomy and durotomy. This is the first section out of three required. This first section is specifically for identifying and marking injection coordinates while the skull is level. These markings will be referenced in the second optional section (Optional 2.1).
Skip this section if you will not be marking injection coordinates for injections after the craniotomy and durotomy. .
Considerations:
For injections within the cranial window, it is important to take the injection depth into consideration. Avoid targeting subcortical brain structures, as deep insertion of the pipette into the brain tissue can increase risk of edema.
Additionally, maintain bregma as the fiducial point. Avoid the use of multiple viruses/pipettes as pipettes are not made identically (variance of up to 0.8mm difference).
Any injection that includes depths deeper than 1mm as being normal to the skull, and depths that are exclusively at target depths of 1mm or less are normal to the cranial window.
Furthermore, for the purposes of the shallower injections, "level" is approximately a roll of 16 degrees and a pitch of 0 degrees.
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject:
Begin at step 8.2.6 "Mark the Injection Site".
Stop before step 8.2.7 "Drilling the Burr Hole".
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".
Ensure injection coordinate is marked as precisely as possible. This is best done with a fine point sharpie mark with a notch or point negative made from the marker pipette tip.
Etch Craniotomy
Using a marker pipette or Bregma stylus attached to the stereotax arm, zero the pipette/stylus directly over Bregma.
Move the pipette/stylus 1mm anterior and 2mm lateral (left) of Bregma. Mark this coordinate with a sharpie or the tip of Dumont forceps on the skull. This will be the center of the window. Zero the pipette/stylus at this new coordinate.
The boundaries of the craniotomy are 1.5mm from the center of the window. Use a sharpie or the tip of Dumont forceps to mark 1.5mm anterior, posterior, left and right of the center of the window.
Remove 3mm coverslip from 70% Ethanol and rinse in two wells of ACSF.
Place the tracer coverslip over the mark for the center of the window and align the edges of the tracer coverslip with the markings made 1.5mm from the center of the window. Use a broken sterile swab in the probe holder to temporarily secure the tracer coverslip in place.
Using Dumont forceps or #11 Scalpel blade make an etch around the 3mm coverslip.
Remove swab and confirm etch with 3mm coverslip.
Using the drill with the FG1/4 or EF4, create a shallow trench over the etch.
Ensure trench is the correct diameter and shape by confirming with the 3mm coverslip.
Using the drill and the FG4, buff the 0.5mm around the outside of the trench.
Using the drill, remove any high points of Metabond that could cause a coverslip to break, or will cause the window to sit incorrectly (not level) within the craniotomy.
Create a Temporary Well
Create a temporary Kwik-Cast well around the craniotomy area.
Cropped image of a Kwik-Cast well around the motor cortex craniotomy etch.
Dispense Kwik-Cast into an empty well within your well plate. Mix and let it sit for 00:01:00 to allow it to slightly stiffen.
Note
Kwik-Cast has an ideal working time within 2-4 minutes of mixing. As it solidifies, it will become increasingly stringy. Do not use once it becomes rubbery.
Once slightly stiff, use the broken end of a swab to create a temporary well around the craniotomy area. It will settle a little, so be sure to allow at least 1.5mm of space away from craniotomy coordinates and areas with excess fur. The temporary well needs to be larger than the craniotomy etch and should allow room to place and seal the coverslip.
Let Kwik-Cast set for about 00:05:00 before proceeding.
Optional (2.1): Align Injection Device for Stereotactic Injections After Craniotomy and Durotomy
20m
Optional 2.1
This optional section accommodates injections that will be performed after the craniotomy and durotomy. This is the second section out of three required. This second section is specifically for aligning the injection device with the desired injection coordinates that were marked in Optional 2. Since the headframe has obscured the coordinate fiducials and a craniotomy will remove the coordinate markings, the injection device needs to be aligned to the precise X and Y (ML and AP) coordinates. After the craniotomy in Optional 2.2, the Z axis (DV) will be manipulated to perform the injections. If there are multiple coordinates, identify one of them and zero the coordinates creating that point as a fiducial to the remaining coordinates.
Skip this section if you will not be marking injection coordinates for injections after the craniotomy and durotomy. .
Attach injection device with loaded pipette onto stereotactic arm and center over injection location. This stereotactic coordinate should be premarked from .
Place the injection device onto the stereotactic arm.
Zero the X and Y and coordinates over marked injection coordinate.
Remove injection device from stereotax arm.
DO NOT move the stereotactic arm out of the way. It will stay in this position during the craniotomy and durotomy.
Note
Nanoject/Iontophoresis pipettes should not be changed after setting up the stereotax arm. The variation in pipette straightness can cause the injection location to move up to 0.8mm.
Perform Craniotomy
40m
Using the drill and the FG1/4 or EF4 return to the trench and drill until the skull is cracked through all the way around.
Note
The drilling process will take 20-40 minutes. Rinse with ACSF to remove blood or debris from the well while drilling. Be aware that soaking the skull in ACSF will cause it to become soft and can cause the drill to take off more layers than anticipated.
Rinse the well using copious amounts of ACSF and sterile gauze/Kimwipes to get rid of debris before removing the skull island.
Place Hemostatic Agent Surgifoam within the well if anticipating bleeds.
Note
The visual cortex craniotomy contains the transverse sinus within the window. It is highly likely that you will require anticipatory Surgifoam ready within the window before removing the skull island.
Add ACSF to the well. Using Dumont forceps, gently pry up the skull at multiple points to ensure the skull island is sufficiently separated.
Note
If skull is still attached (i.e. bone bridge, not cracked), remove ACSF and continue drilling. This is especially crucial on the posterior edge. Rinse before removal.
When the skull island appears ready to be removed (sufficiently detached), use Dumont forceps to carefully remove the skull island, being careful to minimize pressure on the brain (i.e. pressure from skull island or tools).
Stop all bleeds with Hemostatic Agent Surgifoam, ACSF rinses and or sterile Sugi Absorbent Spears. Make sure there is always ACSF covering the brain surface. Ensure all bleeds are stopped as rapidly and efficiently as possible. Excess blood loss can result in mouse death.
Perform a Durotomy
10m
Ensure brain is always covered with a layer of ACSF.
Make the initial incision using a durotomy probe.
Remove dura either by cutting along the bone line or using Dumont forceps to pull flaps over the bone line. Other than around the sinus or vessels connected to dura, no dura should remain within the window (unless connected to sinus or vessel).
Avoid tugging or pulling of the dura as this can cause vessel bleeds, sinus bleeds and/or "piano wire" lacerations.
Use caution around the sagittal and transverse sinuses.
Optional (2.2): Perform Stereotactic Injections Within the Cranial Window
10m
Optional 2.2
This optional section accommodates injections that will be performed after the craniotomy and durotomy. This is the last section out of the three required. This second section is specifically for injecting the desired material at the injection coordinates that were marked in Optional 2.
Skip to next section if not performing stereotactic injections within the cranial window. .
Ensure mouse's skull is level.
Return the mouse to the same angles as performed in .
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject:
Begin at step 8.3 " If Injecting with Nanoject II" or 8.4 " If Injecting with Nanoject III".
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 8.4 "Inject Virus with Iontophoresis"
Stop at step 8.5 "Suturing".
Raise and remove injection device from stereotax arm.
Apply and Secure Coverslip
10m
Rinse the desired coverslip within the two rinse wells of ACSF.
If there are visible air bubbles, rinse again and/or use the ACSF syringe or a sterile swab soaked in ACSF to detach bubbles.
Note
Air bubbles on the coverslip occasionally arise from surface tension when coverslip is being soaked and/or removed from ACSF well. This is to prevent the addition of air into/onto the brain.
Carefully place the coverslip into the craniotomy with Dumont forceps.
Note
If the coverslip is placed too forcefully or dropped, cortex damage may occur.
It is best to have a significant amount of ACSF covering the cortex to cushion its placement.
Load the broken end of a sterile cotton swab into the probe holder and center it above the craniotomy.
Note
The end of the sterile swab should be as flat as possible to ensure even pressure while securing the coverslip. This can be done by cutting the end of the swab and hitting it against a flat and sterile surface.
If accessible, a metal probe with a drop of silicone at the tip can be used as a substitute for a broken sterile swab.
Lower the broken side of the sterile cotton swab very gently onto the center of the coverslip to secure it in the craniotomy, being careful to not crack the coverslip.
Note
Excess pressure on the coverslip can cause detrimental brain compression.
Ideally, the 4mm edge of the coverslip should contact the full perimeter of the craniotomy. This may vary between mice due to skull shape and age.
Remove ACSF from the well with Kimwipes or Sugi Absorbent Spears until the surface tension of the ACSF is held between the 3mm and 4mm edge of the coverslips.
Seal coverslip by placing small drops of Vetbond using an insulin syringe around the coverslip via capillary action, forming a seal between the coverslip and skull surface. Let dry completely. Absorb extra ACSF with Sugi absorbent spears.
Remove Kwik-Cast well. Note any disturbances to the Vetbond seal and reseal if necessary.
1m
Laser Level the Craniotomy
5m
Carefully raise and detach the probe holder from the stereotactic arm.
Watch the Vetbond seal/coverslip closely to identify any ACSF leaks or seal breaks. If leaks occur, reattach and lower the probe, then reapply Vetbond. Proceed to next steps once complete seal is confirmed.
Secure the laser leveling tool in the stereotactic arm and turn laser on.
Align laser onto the coverslip and adjust the pitch and roll of mouse until the laser reflects back up to the center of the laser leveling tool.
Once laser leveled, place digital angle finder on the roll arm of the sterotax and zero. Roll the mouse to -16°.
Pitch the mouse down 3° using the stereotax markings.
Turn off the laser level and remove from stereotax arm.
Secure Headframe
20m
Attach the dovetail clamp to the stereotax arm. Attach the motor cortex headframe to the clamp.
Lower the headframe until there is contact with the skull. Ensure that the headframe is not pushing down on the skull.
Note
The vetbond seal can often break during the placement of the headframe. Before proceeding to the next step, check for compromised areas and re-vetbond if necessary.
Note
Lowering the headframe too far down onto the mouse's skull can potentially crack a cranial suture line. If this occurs, your headframe will not attach to the skull due to Cerebrospinal fluid leakage.
Prepare and apply the first round of Metabond.
Apply Metabond inside the headframe and around the outside where the skull is easy accessible around the stereotactic frame. Place Metabond so that the majority of the skull within the well remains exposed. Let dry ( ~ 00:05:00 ).
Turn isoflurane off, remove earbars, and loosen the nose cone slightly.
Carefully detach the headframe from the earbar headclamp and/or earbars.
Prepare another Metabond tray.
Pull back nose cone completely, and remove the mouse from bite bar. Either lie the mouse on the heating pad or hold mouse in hand.
Apply Metabond to the outside of the headframe to cover all exposed muscle. Extend Metabond to 1-2mm past the exposed muscle and tissue to create a seal.
Note
Be as efficient and quick as possible to ensure mouse stays anesthetized during this step.
Note
Ensure Metabond is not left too close to eye and does not have any sharp points. This is to avoid any irritation to the mouse.
Using the 0° earbar headframe clamp, return mouse back onto the stereotax. Ensure headframe is secured in place by ear bar clamp.
Note
Be as efficient and quick as possible to ensure mouse stays anesthetized during this step.
Place headframe plate into the clamp, nose cone to nose, and turn isoflurane on.
Replace saran wrap over mouse body.
Recover Mouse and Takedown
5m
Remove mouse from stereotactic instrument base once Metabond is fully dry.
Turn off isoflurane.
Detach headframe 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.
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 procedures.
Protocol references
Allen Institute for Brain Science. (2023). Stereotaxic Injection by Nanoject Protocol V.6. protocols.io. dx.doi.org/10.17504/protocols.io.bp2l6nr7kgqe/v6
Allen Institute for Brain Science. (2024). Stereotaxic Injection by Iontophoresis V.6. protocols.io. dx.doi.org/10.17504/protocols.io.14egn8ewzg5d/v6