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🔬 Nanoinjection Protocol using Nanoject III

This protocol details the materials and steps for performing microinjections using the Nanoject III system, typically for injecting solutions like viruses or dyes into biological samples (e.g., embryos, specific brain regions).

📦 Materials Required

| Item | | :--- | :--- | | Nanoject III | | Injection Pipette | Capillary glass needle for injection. | | Solution | The virus or dye to be injected. | | Mineral Oil | Used for filling the pipette and preventing air bubbles. | | Mineral Oil Syringe | Used for the initial oil fill into the Injection Pipette. |

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⚙️ Procedure Steps

1. Pipette Filling and Preparation

1. Fill the pipette with the mineral oil syringe slowly, without allowing for air bubbles.
2. Slowly inject mineral oil until you see it on the tip, then gently retract the syringe while still injecting to maintain the fill.

2. Nanoject Setup

3. Insert the injection pipette into the mounted Nanoject. This is done by unscrewing the tip slightly, inserting the pipette until resistance, then tightening the screw.
4. Turn the Nanoject III machine on and press the PROGRAM button.
5. Press the EMPTY button and observe the Nanoject needle entering the pipette.

3. Loading the Injection Solution

6. Place the tip of the mounted pipette into the solution (virus or dye) you need. The solution will most likely be in a 100 μL tube. Using some of the gum that the injection pipettes are used for transport, hold the tube underneath the Nanoject. This will help with alignment. Use microscope to ensure that the needle is in the solution.
7. Press the FILL button on the Nanoject. The machine will fill the pipette automatically.
   • Crucial Air Bubble Check: Ensure there are no air bubbles in the solution column.
   • To prevent air bubbles, the fill rate should be set to 20 nL/second. Faster rates increase the likelihood of air bubbles.

4. Running the Injection Program

9. Standard Injection Settings (Example for ACC):
   • Volume: 50 nL
   • Cycles: 3 cycles
   • Interval: 60 seconds between each cycle
   • Rate: 20 nL/sec
   • These settings are configured under the PROGRAMS menu.
10. To create a new program, press NEW. Once settings are confirmed, press RUN to begin the injection.

5. Post-Injection Cleanup

11. To safely remove the injection pipette, press the HOME button before unscrewing the pipette. This retracts the internal needle.
12. Remove the pipette by unscrewing the holder on the Nanoject.