To begin, insert a micropipette into the micropipette puller and pull a microinjection needle. Align the needle tip with the divisions of the stage micrometer under a dissecting scope. With the micrometer as a measurement guide, use a sharpened pair of forceps to break the needle to a bore size slightly larger than the cell's diameter.
Using a 50-milliliter syringe equipped with a pipette filler, completely fill the needle with the mineral oil. On the transplant apparatus, turn the three-way stopcock so that its long arm faces the microsyringe pump. Depress the reservoir syringe plunger to flush all air bubbles from the hydraulic line.
Maintaining light pressure on the plunger, turn the three-way stopcock so that the long arm now faces the reservoir syringe. Then insert the needle into the holder without introducing air bubbles. Adjust the needle so that it faces directly to the left at an angle of 10 to 15 degrees from the horizontal.
Using the coarse and fine micromanipulators, maneuver the needle tip under the microscope objective to bring it into focus. If the liquid is flowing into or out of the needle tip, adjust the pressure using the syringe pump until a stable meniscus between the oil and RPT is observed. If an oil bubble remains, move the needle to the right to withdraw its tip from the RPT, then back to the left for repositioning.
Move the stage to the right until the donor animal is brought back into view. Recenter and focus on the cells to be transplanted, aligning the needle with the cells just outside the animal. Next, insert the needle into the donor animal, and if necessary, restabilize the oil meniscus in the needle tip with the microsyringe pump.
Position the needle tip against the cells of interest and apply gentle suction with the microsyringe pump. After taking adequate cells, remove the needle from the donor and restabilize the material in the needle tip. Reposition the stage to bring the first host animal into view/Center and focus on the area in which the cells are to be placed and align the needle with this region just outside the animal.
Then insert the needle into the host animal and restabilize the oil meniscus in the needle tip. Position the needle tip in the deposition site and apply gentle pressure with the microsyringe pump until the correct number of donor cells are released from the needle. Finally, remove the needle from the host.
At 12 hours post-transplantation, a donor neuron was successfully observed in the anterior region of the host's vagus motor nucleus, showing signs of healthy neurite protrusions. By two days post-transplantation, the transplanted neuron not only remained correctly positioned but had also begun extending a new axon toward the fourth pharyngeal arch, indicating successful integration within the host.
