Apr 02, 2025

Public workspaceMouse stereotaxic surgeries for Projection-TAGs

DOI
dx.doi.org/10.17504/protocols.io.5qpvo9ejxv4o/v1
  • 1Washington University in St. Louis
  • Projection-TAGs
Lite Yang
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo9ejxv4o/v1
Protocol Citation: Fang Liu, Hannah Hahm, Yufen Zhang, Lite Yang, Vijay Samineni 2025. Mouse stereotaxic surgeries for Projection-TAGs. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9ejxv4o/v1
Manuscript citation:
Yang L, Liu F, Hahm H, Okuda T… MR, Samineni VK. Projection-TAGs enable multiplex projection tracing and multi-modal profiling of projection neurons. bioRxiv [Preprint]. 2024 Apr 28:2024.04.24.590975.
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 05, 2025
Last Modified: April 02, 2025
Protocol Integer ID: 123816
Keywords: Stereotaxic injection, Multiplex projection tracing, Projection-TAGs, Neural circuit
Funders Acknowledgements:
National Institute of Diabetes and Digestive and Kidney Diseases
Grant ID: 5R01DK128475-04
Abstract
This protocol describes the process of performing stereotaxic surgeries on mouse for multiple Projection-TAG injections.

For more Projection-TAGs protocols, please visit: https://www.protocols.io/workspaces/projection-tags

Materials
Equipment
  1. Stereotaxic frame (David Kopf, Model 940)
  2. High-speed micro-drill (RWD Life Science, 78001)
  3. Nanoject II Auto Injector (Drummond Scientific, 13-681-455)


Reagents
  1. Isoflurane (DailyMED, NDC 66794-017-25)
  2. Corn oil (Sigma-Aldrich, C8267)
  3. 5-0 nylon sutures (AD Surgical, S-N518R13)
  4. Betadine solution (McKesson, 67618015017)
  5. Cotton swab (Dealmed/Amazon, 782010)
  6. Triangle sponge points (Preferred Products, KT- 30761)
  7. Eye ointment (Bausch+Lomb/Amazon)
  8. 0.9% Saline
  9. 70% Ethanol
  10. Scissors
  11. Forceps
  12. Hemostat
Before start
Please carefully read the critical steps in the protocol as they are specifically optimized for Projection-TAG experiment.

The following steps should be done the day prior to surgery.

  1. Autoclave and sterilize surgery tools. If surgery will be performed on multiple animals in a single surgery session, use hot bead sterilizer for the appropriate temperature and time to re-sterilize tools.
  2. Weigh and shave experimental mice.
  3. Pull injection needles using glass capillaries and a micropipette puller.
Pre-Surgery
Pre-Surgery
Set Up
Gather necessary tools: corn oil, 70% ethanol, betadine, saline, cotton swab, triangle sponge points, eye ointment, scissors, forceps, hemostat, 5-0 nylon sutures, scalpel, injector.
Obtain virus aliquots from the -80C freezer and dilute with saline to the desired titer. Place the virus in an ice bucket.
Turn on the bead sterilizer and light.
Turn on the injection device.
Turn on heating pad to medium.
Place your sterile surgical tools (forceps, scalpel, small scissors, hemostat, and surgical clips)
Turn on isoflurane chamber and set isoflurane concentration to 4%
Injector Preparation
Trim end of injection needle and fill with corn oil using 27G needle.
Assemble the injector.
Clean the needle tip with a cotton swab.
Load virus with additional 20% volume (withdraw 60nl/s).
Test the pipet by infusing 30nl of virus (Infuse 1n/s).
If doing multiple injectors are available, next virus/injector can be prepared while the last injection is ongoing. If doing multiple injections using the same injector, make sure to wipe injector needle with 70% ethanol and change the glass needle for different viruses. To avoid cross-contamination, do not reuse the needle for different viruses.
Critical
Anesthesia/mouse preparation
Place mouse in isoflurane chamber with 4% isoflurane.
Once mouse is fully unconscious and its respiratory rate slows to ~1/s, place it in stereotaxic frame (David Kopf Instruments) fitted with a gas-anesthesia mask.
When the mouse does not respond to toe pinch, adjust isoflurane concentration to 1-1.5% for maintenance.
Mount animal by placing ear bars along ear canals adjacent to meatus tissue of the ear. Ensure head is firmly ensured and level.
Apply topical eye ointment bilaterally to prevent corneal damage
Confirm there is no toe pinch response. 
Continuously monitor breathing rates and depth of anesthesia throughout the surgery.
Note: Respiratory depression may lead to death of the experimental mouse during surgery. If the breath becomes deep and slow (slower than once per three seconds), decrease isoflurane concentration by 0.25% and/or adjust the isoflurane flow rate to prevent further respiratory depression.
Critical
Surgery (Intracranial)
Surgery (Intracranial)
Clean and sterilize scalp with betadine solution.
Apply intradermal injection of bupivacaine (8mg/kg) where incision will be made.
Make a rostral-caudal incision along the midline of the scalp to expose lambda and bregma points of the skull.
Gently clean cranial surface with scalpel and wipe with 70% ethanol cotton swabs.
Attach the drill to the manipulator arm of stereotaxic frame.
Balance the brain.
Go to bregma with the drill and zero the coordinates.
Go to lambda and check the dorsal-ventral (D/V) coordinate. Adjust head position until bregma and lambda are level within ±0.03 mm difference in D/V coordinate.
To level the left and right sides of the brain, move needle ± 2.0 mm from midline and adjust head position until D/V coordinate of the two points are within ±0.03 mm difference.
Check the D/V coordinates on Bregma and Lambda again.
Go to Bregma and zero the coordinates.
Go to the coordinates of area of interest.  
Drill hole directly above the brain region of interest using appropriate drill bit and slowly adjusting DV.
Before injecting, make sure the dura at the injection site is punctured, which can be done by using a 32G needle whose tip has been bent to pierce the drill hole. A small bead of blood will typically appear in the hole.
Gently clean up the drill hole using the tip-bent needle and remove blood with triangle sponge points.
Remove the drill and load the injector on the stereotaxic machine.
Go back to bregma with the needle and zero the coordinates.
Go to the coordinates of area of interest.  
Insert needle into brain at the speed of 0.1 mm/s until going past the desired D/V position by 0.05 mm.
Retract the needle to the desired D/V position.
Slowly inject virus (Infuse 1n/s).
Note: monitor the volume of the virus to identify potential needle clog and ensure successful injection.
Upon injection completion, wait in place for at least 8 minutes to allow diffusion of virus.
Withdraw the injector by 0.05 mm and wait in place for additional 2 minutes.
Slowly withdraw the injector at the speed of 0.1 mm/s.

Repeat steps 16-23 for additional injections.
After completion of all injections, suture with 5-0 nylon sutures.
Apply topical antibiotics to incision site.
Surgery (Intraspinal)
Surgery (Intraspinal)
Note: If forming intraspinal and intracranial surgeries on the same mice, the surgeries should be done separately with enough interval time for the animals to recover.
Critical
Disinfect skin with three alternating washes with alcohol and betadine.
Apply intradermal injection of bupivacaine (8mg/kg) where incision will be made.
Cut a midline incision from the level of the iliac crest to the highest point of the spine.
Identify the targeted spinal cord segment and secure one spinal adaptor on the stereotaxic device while keeping the other one loosely attached. Then use the forceps with teeth to place the vertebra between the spinal adaptors, tighten the second adaptor to hold the mice partly suspended.
Bluntly dissect the muscles surrounding the spine to expose the vertebrae.
Carefully use the drill to cut and remove the vertebrae and to exposure the spinal cord.
Insert needle into spinal cord below the dura, taking care to avoid the posterior spinal arteries. Set the posterior spinal arteries ML as zero.
Go to the following coordinate by slowly inserting the needle at the speed of 0.1 mm/s.
For the dorsal horn injection, ML: 0.3-0.5 mm, DV: 0.4/0.2 mm, 250 nl virus/site
For the sacral DGC injection, ML: 0.1 mm with injector at 10-degree angle, DV: 0.54-0.58 mm, 5 injection sites with 80-100 nl virus/site)
Slowly inject virus (Infuse 1n/s).
Note: monitor the volume of the virus to identify potential needle clog and ensure successful injection.
Upon injection completion, wait in place for at least 8 minutes to allow diffusion of virus.
Withdraw the injector by 0.05 mm and wait in place for additional 2 minutes.
Slowly withdraw the injector at the speed of 0.1 mm/s.
Repeat steps 28-39 for additional injections.
After completion of all injections, close latissimus dorsi and skin, suture with 5-0 nylon sutures.
Apply topical antibiotics to incision site.
Post-operative Care
Post-operative Care
Inject 0.5 ml of saline subcutaneously to re-hydrate the mouse.
Inject Buprenorphine-SR (0.5-1.0 mg/kg) subcutaneously.
Remove the mouse from isoflurane and monitor until mouse full recovers.
Provide carprofen (50 mg/kg) ad libitum in water for 3-5 days post procedure and monitor mouse for 4 days post procedure.