Stereotaxic Injection Mouse Surgery

Materials

1. SomnoSuite
2. Meloxicam (1:10 concentration)
3. Stereotactic apparatus
4. 70% ethanol
5. Heating pad
6. Isoflurane
7. Standard U-Frame Stereotaxic instrument
8. Suturing kit
9. Scalpel, scissors, pointy and round forceps
10. Injection needle
11. Mineral oil
12. Coordinate system
13. Topical eye ointment
14. Microscope
15. Iodine
16. Clean mouse cage
17. Paper towels
18. Drill with small drill tip
19. Sharpie

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Procedure

1. Make Injection Pipettes Before Setting Up

a. Retrieve the green box in the patch clamp room; use the smaller diameter pipettes (silver cap).
b. Insert the pipette into the groove with equal length on both sides. Lift the bottom with fingers and tighten the knobs.
c. Press Step 1, then Start to heat-separate the pipette. Wait for the coil to cool before removing.
d. Cut the pipette near the middle. Dispose of the top portion in sharps; place the bottom portion onto the petri plate with white gum.

2. Prepare the Mineral Oil Pipette

a. Fill the pipette with mineral oil using the syringe, avoiding air bubbles.
b. Insert the injection pipette into the mounted Nanoject by loosening the tip and adjusting slowly.
c. Turn on the Nanoject III, press Program, then Empty to allow the Nanoject needle to enter the pipette.
d. Add virus or dye by pressing Fill. Ensure no air bubbles.

3. Set Up the Stereotaxic Apparatus

Set up ear plates, drill, heat pad, SomnoSuite anesthetic system, meloxicam, saline, and tools.

a. Meloxicam preparation: 1:10 dilution with saline.

• Example: Stock is 5 mg/mL → add 5 μL stock + 25 μL saline.
  b. SomnoSuite note:
• Ensure clamps match color coding.
• Release notches when switching lines; never close all notches at once.

4. Induce Anesthesia

Induce mice in chamber at 5% isoflurane using SomnoSuite.

a. Weigh the mouse after anesthesia onset.
b. Check anesthesia depth by firmly pinching the hind paw nail. Remove hind paw nails after induction. Continue if reflex persists.
c. Apply topical eye ointment generously; roll ointment onto each eye.

5. Position the Mouse in the Stereotaxic Frame

Insert the mouth over the mouthpiece (not too tight).
Use pointy forceps gently under teeth to open mouth.
Pull tail back to ensure alignment.

6. Secure the Head

Lightly tighten side pieces around the zygomatic bone.
Set anesthesia maintenance to 1–2%.

7. Inject Meloxicam

Inject meloxicam (1:10) subcutaneously in the lower trunk.
Create a skin tent and insert needle at the tent base. Dose according to weight.

8. Prepare Surgical Field

Shave skull with a razor.
Clean with iodine, then 70% ethanol, in circular motions.
Repeat cleaning 3×.

9. Drape the Mouse

Cover the head with a drape and cut an opening to expose the skull.

10. Make the Incision

Make a midline incision to reveal bregma and lambda.

11. Level the Skull

Center drill at bregma and align skull.

a. Level left–right by adjusting side ear bars; repeatedly return to bregma to check.
b. Level anterior–posterior axis using bregma as reference.

12. Move to Desired Coordinates

Use the Mouse Brain Atlas.

Examples:

• Ventral Pallidum: AP +0.6, ML +1.5, DV –4.6
• MD: AP –1.7, ML +0.3, DV –3.43
• ACC: AP –0.6, ML +0.3, DV –1.25

13. Drill Holes

Drill bilaterally at target coordinates—avoid drilling too deep.
Recalibrate at bregma after each drill site.

14. Replace Drill With Nanoject

Insert Nanoject.
Mark the top of the fluid column with a Sharpie.

15. Perform the Injection

Navigate to target coordinates and lower to DV depth.

a. Injection volume depends on experimental protocol.
b. Nanoject III settings are under Preferences.

16. Close the Surgical Site

Suture or apply surgical glue by pinching skin edges together and applying a small amount of glue.

17. Post-Operative Care

Place mouse in a clean cage.

a. Place food and water on the floor.
b. House mouse individually to prevent aggression.
c. If the mouse appears dehydrated, distressed, or hyperactive, administer saline bolus and contact DCM veterinary staff for antibiotics if needed.