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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
- Preparing the microinjection syringe and microinjector for injection:
- Lock each gastight microinjection syringe into the rack of the microinjection pump.
- Place the tip of the gastight syringe into the sterile water contained in a small weigh boat and gently flutter the plunger to lubricate the internal wire. Evenly pull the plunger to the backstop on the pump to fill it with water.
- Slide the PE20 tubing that is connected to the microinjector onto a 26 G (brown hub) needle attached to a 1 ml syringe that is filled with sterile water. Spray sterile water through the microinjector, and inspect spray pattern and distance.
NOTE: Water should spray very far and straight out of the microinjector tip. If the stream is not straight, the injection location will not be correct. - Place the sterilized microinjector onto a sterile field.
- Slide the tubing off of the needle, keeping the microinjector tubing upright to prevent water from dripping out while also cutting the tubing below the area damaged by the needle.
- Push the microinjection syringe plunger slightly forward to create a droplet on the end of the needle and slide the water-filled PE20 tubing (that is connected to the microinjector) onto the needle.
NOTE: This will create a liquid-to-liquid connection that will allow appropriate backpressure that could dislodge a clog that could form at the tip of the microinjector. - Push the plunger completely forward to prepare for sample loading and ensure that no trace ethanol remains in the microinjector.
- Check the setup for leaks by touching the water droplet that has formed at the tip of the microinjector to a clean lab wipe multiple times.
- NOTE: When touching the lab wipe, only one wet spot should form. If there are more drops, there is a leak in the system.
- Avoid leaks by controlling super glue application and by trimming the PE20 tubing with sharp scissors or a razor blade any time that the PE20 tubing is removed from any connection.
- Pull the syringe plunger back 0.2 μl; creating a bubble between the sterile water in the PE20 tubing/ microinjector and the solution to be injected.
- NOTE: This bubble prevents dilution of the injectate, contamination of the water, and allows for monitoring of the injection process since it will move during the injection. A single, solid bubble should be produced.
- Place the microinjector into the reagent to be injected and slowly pull the syringe plunger to the backstop.
- Preparing an animal for injection:
- Hold the rat’s chest against the experimenter’s chest. Clean the area around the cannulae with a cotton applicator soaked in 70% ethanol (v/v).
- NOTE: With proper handling, mice can be gently restrained in a cupped hand. Otherwise, scruffing may be required.
- Remove obturators using small forceps and place them into the weigh boat with 70% ethanol (v/v).
- Performing injections:
- Place the filled microinjector into the cannula. Press start on the microinjection pump and monitor bubble movement.
- NOTE: It may be necessary to apply light pressure to ensure that microinjectors do not lift. The bubble should uniformly advance and fully enter into the microinjector.
- Remove the microinjectors from the cannula and replace obturators, once the injection is complete and the post-injection diffusion period has passed.
- NOTE: Typical post-injection diffusion times are 2 – 4 min for pharmacological reagents and 2 – 10 min for viruses. Leaving the microinjector in during the post-infusion period prevents the injectate from refluxing back up the cannulae rather than diffusing into the tissue. Place obturators on the side of the ethanol-filled weigh boat to allow the ethanol to drain before reinserting into the cannula.