Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

My research focuses on the neurodevelopment of the cochlea. Specifically, I aim to uncover the underlying mechanisms of SNAPS formation within the cochlea and investigate the connection between Alzheimer's disease and hearing loss. Live imaging and genetic tools are improving our ability to study cochlear structures and neural circuits in greater detail, providing deeper insights into auditor system resilience and dysfunction. By publishing this protocol, we want to help researchers who are new to auditor science, especially those who haven't worked with cochlea before. This guide explains step by step how to prepare inner ear samples from embryonic, newborn and adult mice. Our aim is to make inner ear experiments easier, and ensure the results are reliable and consistent. Cross-section immunostaining has several advantages over other methods, like whole-mount immunostaining. It preserves proteins and nucleic acids well, and creates thin, nearly 2D sections, which makes measurements more accurate and consistent. This method also keeps all cochlear cell types intact and visible, improving the cult of immunostaining. Unlike whole-mount preparations, cross-sectioning preserves important structures, like the spiral ligament, stria vascularis, Reissner's membrane and tectorial membrane.

[Instructor] To begin, place the juvenile euthanized mouse on a surgical platform. Using fine dissection scissors, carefully make a midline incision along the scalp, starting from the neck and extending toward the snout. Next, place the bottom blade of the scissors at the foramen magnum, and the top blade at the cranium. Cut the top half of the cranium up to the nose to complete the second cut. Back the scissors out of the partially incised head. Place the bottom blade of the scissors at the bottom of the cranium, and the top blade at the foramen magnum. Cut the bottom half of the head up to the nose to complete the third cut. Using fine forceps and scissors, carefully detach any soft tissue surrounding the temporal bone, including the brain, muscles and connective tissue. Next, place the mouse half heads into wells of a 24-well plate containing PBS. Replace the PBS with 4% paraformaldehyde solution for fixation and incubate for 45 minutes at room temperature. After fixation, rinse the tissue three times for five to 10 minutes with PBS. Locate the temporal bone or inner ear capsule within the mouse half head. Using fine forceps, carefully detach the soft tissue surrounding the inner ear capsule. Once loosened, cut the tissue around the inner ear capsule. Then apply gentle pressure to the area surrounding the inner ear capsule using forceps to free it completely. Store the inner ear samples in PBS at four degrees celsius. To begin, place the adult euthanized mouse on a surgical platform. Using sharp dissection scissors, make a midline incision along the scalp, starting from the neck and extending toward the snout. Fold back the skin to expose the skull during the first cut. Place the bottom blade of the scissors at the foramen magnum, and the top blade at the cranium. Cut the top half of the cranium up to the nose to complete the second cut. Back the scissors out of the partially incised head. Position the bottom blade at the cranium base, and the top blade at the foramen magnum. Cut the bottom half of the head up to the nose along the midline, avoiding the inner ears. For each half head, use forceps and scissors to detach soft tissue surrounding the temporal bone, including the brain, muscles and connective tissue. Next, reverse curl the skull tissue with fingers and use the thumb to apply gentle pressure to the underside of the temporal bone. Gradually loosen the bone from its surrounding attachments. Once the inner ear capsule is removed, rinse it thoroughly with PBS. Place a Sylgard dish filled with 4% paraformaldehyde under the microscope. Position a single inner ear capsule in the dish. Examine the inner ear for any attached soft tissue or bone, and gently remove it. Identify the oval window and gently remove the stapes using forceps. Next, using fine forceps, open a small hole in the apex of the cochlea. Using a P20 pipette filled with 4% paraformaldehyde, flush the cochlea to ensure fluids, like dilute blood or endolymph, exit through the oval window. Transfer the fixed cochlea into a 24-well plate filled with 4% paraformaldehyde. Mark the time and incubate for 30 to 60 minutes with gentle agitation using a rocker or nutator. After incubation, rinse the sample three times for five to 10 minutes with PBS. Following the final rinse, place the cochlea in 1.25-millimolar EDTA and allow it to rock for two to three days at four degrees celsius. After EDTA treatment, rinse the cochlea three times for five minutes with PBS. To begin, place the dissected inner ear capsule in 10% sucrose solution, and incubate at room temperature for two hours. After incubation, replace the 10% sucrose solution with 20% sucrose and incubate for another two hours. Then replace the 20% sucrose solution with 30% sucrose and incubate the sample overnight at four degrees celsius. The next day, remove half of the 30% sucrose solution. Fill the space with optimal cutting temperature or OCT compound and let the sample rock for 30 minutes to two hours. Transfer the samples into labeled cryomolds filled with OCT compound. Check the orientation of the cochlea in the embedded cryoblock. Ensure the inner ear;s concave side faces the cryomold's narrow sides for standard cochlear cross-sections. Next, place dry ice in a small container and add five to 10 milliliters of dimethylbutane. Place the cryomolds containing OCT and the sample on the bubbling dry ice to rapidly freeze the block. Transfer the cryoblocks to the chamber of a cryostat pre-chilled to minus 20 degrees celsius. Allow the samples to equilibrate for 30 to 60 minutes. Using a pencil or pen, mark the side of the cryoblock corresponding to the cochlear position to ensure proper orientation for sectioning. Next, add a small pool of OCT to the chuck and place the cryo block on it, ensuring the broad side without the pencil mark is facing down. Place the chuck containing the OCT and the sample in the chilled cryostat chamber, allowing the OCT to freeze completely. After a few minutes, position the chuck with the sample onto the chuck holder with the pencil mark pointing to the right or left. On a cryostat, trim the block using a 40-micrometer setting until the tissue becomes apparent. Once the tissue is apparent, harvest a 12-micrometer section using slides designed for cryosectioning. Check the section location under a microscope. If approaching cochlear turns, continue harvesting 12-micrometer sections, periodically checking the position until all the cochlear turns have been sectioned.