Cochlear surface preparations allow visualization of the entire lens of the organ of Corti using immunohistochemistry and confocal imaging. It has been broadly used for investigation of specific cochlear pathologies of interest. We will modify the cochlear microdissection method.
The main advantage of this technique is the adherence of a piece of cochlear epithelium to 10 millimeter round coverslips for the immunolabeling procedure while avoiding tissue loss during the multiple washing steps. In addition cochlear pathologies, cochlear surface preparation can also be utilized for the assessment expression and hair cell regeneration. Basic microscope skills are required for this technique and visual demonstration of the method can help to ensure that each step is performed correctly.
Demonstrating the procedure will be Qiaojun Fang, a graduate student from my laboratory. For temporal bone extraction, immediately post-mortem pull the skin anteriorly to expose the skull bone and use scissors to cut the bone from the posterior aspect forward along the center line of the skull. Use forceps to remove the brain tissue before using the thumb and index finger to manually remove the temporal bones from mice three months of age or older.
Place the bones into a 30 millimeter diameter Petri dish containing fresh ice cold 4%PFA and PBS under a dissection microscope and remove the stapes from the oval window. Puncture the round window membrane with number five forceps and use a 27 gauge needle attached to a one milliliter syringe containing fresh 4%PFA to make a small hole into the apex of the cochlea. Gently and slowly perfuse the cochlea with a fixative solution via the round and oval windows until the solution washes out of the small hole at the apex.
Then transfer up to two perfused cochleae into individual 20 milliliter scintillation vials containing 10 milliliters of 4%PFA per vial and gently agitate the samples for two hours at room temperature, followed by overnight storage at four degrees Celsius on a rotator. The next morning, wash the cochleae with three five-minute washes with fresh PBS per wash. After the last wash, add 20 milliliters of 4%EDTA to each vial and rotate the samples for 48 hours at four degrees Celsius with gentle agitation.
At the end of the incubation, touch the bony vestibular portion of each cochlea with forceps to assess the elasticity of the samples. If the bones are elastic rather than firm, the cochlea have been decalcified. Replace the EDTA for each sample with fresh PBS.
For microdissection of the cochlear epithelium, grasp the vestibular portion of the temporal bone with forceps and use a scalpel to cut the apical turn at a 45 degree angle. Cut vertically along the faint line between the round window and the oval window to separate the cochlea from the vestibular portion and orient the cochlear portion with the basal turn toward the bottom and middle turns toward the top of the Petri dish. From this position, cut the bony capsule and lateral wall of the middle turn toward the end from which the apical section was removed and continue cutting to completely separate the middle portion from the basal and hook regions.
Placing the basal and hook portion with the basilar membrane site oriented toward the bottom of the dish, vertically cut the medialis off of the hook region to remove the medialis and cut the basal and hook regions to separate the hook portion. To avoid distortion of the tissue, cut the medialis off of the hook region before separation of the basal turn and hook region. Cut away the relatively large portions of bony capsule and lateral wall tissue of the middle turn and holding the lateral wall with forceps to align the bony capsule and lateral wall with the bottom of the Petri dish, cut these tissues from the basilar membrane side.
Next, flatten the specimen to orient the sensory hair cell surface side up and trim away the rest of the bony capsule and lateral wall. Then use forceps to remove the tectorial membrane to completely separate the middle region and remove the bony capsule, lateral wall tissue, and tectorial regions of the remaining turns as just demonstrated. When all of the cochlear turns have been dissected, spread 0.5 microliters of cell and tissue adhesive onto the center of one round 10 millimeter coverslip and allow the adhesive to dry for three to five minutes at room temperature.
Place the dried coverslips into the Petri dish and stick all four pieces of each sensory epithelium sample onto one coverslip. Then use forceps to grasp one edge of each coverslip to transfer the specimens into individual wells of a four-well cell culture dish for immunolabeling. For immunolabeling of surface cochlear synapse preparations, wash each sensory epithelium three times for five minutes in fresh PBS per wash, followed by the treatment of each sample with two milliliters of 2%non-ionic surfactant for 30 minutes at room temperature on a rotator.
At the end of the incubation, aspirate the surfactant solution from each well and block any nonspecific binding with 100 microliters of blocking solution per well for one hour on the rotator with gentle agitation at room temperature. At the end of the blocking incubation, wash the samples three times with PBS as demonstrated under gentle agitation before labeling each epithelium with 100 microliters of the primary antibodies of interest for 24 hours at 37 degrees Celsius protected from light. The next day, wash the samples three times with fresh PBS and gentle agitation per wash and label the specimens with 100 microliters of the appropriate secondary antibodies of interest for two hours at 37 degrees Celsius protected from light.
At the end of the incubation, wash the samples with three five-minute washes in fresh PBS per wash before placing each coverslip onto individual glass microscope slides with the samples facing up. Next, carefully add eight microliters of an appropriate mounting medium into the center of each coverslip and use forceps to mount another 10 millimeter coverslip onto each sample. Then seal the sides of each coverslip sandwich with clear nail polish, place the samples into a cardboard slide folder, and store the slides at four degrees Celsius.
Although the dissection of adult mouse cochleae for surface preparations is not simple, researchers new to the technique should be able to learn the method after practicing with 10 to 15 ears. Immunolabeling of surface preparations of untreated 10 to 12-week-old CBAJ mice with CTBP2 and GluA2 demonstrates that both presynaptic ribbons and postsynaptic terminals respectively are located below the inner hair cell nuclei and are juxtaposed indicating functional synapses. Immunolabeling for myosin 7A and counterstaining with phalloidin and DAPI reveals the presence of sensory hair cells including the outer hair cells and inner hair cells and their nuclei.
Immunolabeling for myosin 7A and counterstaining with phalloidin shows no differences in immunoreactivity or uniformity with or without the use of cell and tissue adhesive. In addition, scanning electron microscopy of surface preparations from untreated six to eight-week-old C57 black 6 mice demonstrate a well-organized V-shaped stereocilia of outer hair cells in three rows of the sample. Remember to fill the cochlear with fixative solution gently and slowly through the round and oval windows until the solution washes out of the small hole at the apex.
Before separating the hook region from the basal turn, take care to cut the medialis off of the hook region to facilitate the removal of the medialis.
