Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear
筆記録
Scoop out the brain of the euthanized mouse using closed surgical scissors. Sever and remove the cranial nerve to completely detach the brain from the skull. Starting at the back of the head, pull the clear membranous material off with forceps, and then cut the top of the skull out using surgical scissors.
Remove the excess tissue from the back of the head and neck, to make the dissection area and otic capsule, cleaner and easier to access. Transfer the tissue to a second Petri dish with fresh L-15 solution. Then, locate the otic capsule and the auditory, superior vestibular, and inferior vestibular nerves. Separate the superior and inferior ganglia using small spring scissors, and cut away the auditory nerve.
Using a scalpel, gently shave off the bony ridge to weaken the bony area under which the nerve dives into the bony capsule. Carefully remove the debris with a scalpel, exposing the entire swollen portion of the ganglion.
Use the fine spring scissors to cut and separate the ganglion from the peripheral nerve branch that is diving toward the utricle. Remove the superior ganglion using fine forceps, and transfer it to a 35-millimeter Petri dish with fresh L-15 solution.
After preheating the enzyme solution, pour the enzyme mixture into a 35-millimeter Petri dish, and place it in a 37 degrees Celsius incubator for 10 to 15 minutes. Clean the ganglion using fine forceps and small spring scissors by removing bone, excess tissue, nerve fibers, and any other superfluous structures. Take care to minimize the removal of any ganglionic tissue.
Transfer the cleaned ganglia into the preheated enzyme solution, and place it back in the incubator for 10 to 40 minutes. Then, transfer the ganglia to the 35-millimeter Petri dish with fresh L-15 solution.
After two to three minutes, transfer the ganglia to another 35-millimeter Petri dish filled with filtered culture media. Transfer approximately 150 microliters of filtered culture media onto a coated glass bottom dish. Then, transfer the desired number of ganglia to the coverslip.
Draw a small amount of culture media from the culture dish, and rinse the trituration pipette with medium to prevent the tissue from sticking to the sides of the glass pipette. Triturate by gently and repeatedly passing the tissue through the pipette until the ganglia are sufficiently dissociated.
After five minutes, check under a light microscope to see if the cells have settled on the coverslip. Carefully place a culture dish into a 37 degrees Celsius incubator for 12 to 24 hours.
