This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.

Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.

Included in our methods are special considerations made for the cochlea given its
shape and structure. Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

Understanding auditory development and function is crucial for characterizing and addressing different forms of hearing loss. Risk factors for hearing loss are many and include diabetes^1,2, hypertension^3,4,5, autoimmune diseases^6,7, bacterial meningitis^8,9, and several neurodegenerative disorders^10,11,12,13. Additionally, certain drugs, such as some antibiotics and chemotherapy medications, can also negatively impact hearing¹⁴. Beyond the immediate challenges of auditory impairment, hearing loss can negatively impact cognitive function and increase anxiety and stress, which can correlate with cognitive impairment and mental health decline^11,15,16. By publishing this protocol, we aim to ease any barriers to entering auditory research, particularly for those without prior experience working with the cochlea. This guide explains how to prepare inner ear samples from embryonic, juvenile (neonatal), and adult mice. The purpose of this protocol is to provide a straightforward approach to inner ear tissue processing, ensuring reproducibility and consistency across experiments. Section immunostaining is one of the best methods for examining different cell types in the context of the whole cochlea; in other circumstances, whole-mount immunostaining may be more advantageous (e.g., examining hair cell stereocilia morphology or protein localization).

The rationale for this stems from challenges working with the mouse cochlea. Its small size, unique coil, and delicate structure¹⁷ require specialized techniques for accurate dissection, fixation, embedding, cryosectioning, and immunostaining. The spiral shape of the cochlea means that embedding requires careful attention to orientation to ensure that each turn of the cochlea is preserved, which is crucial for obtaining consistent sections. Additionally, as the cochlea matures, it undergoes ossification¹⁸, meaning that it gradually transforms into bone, which can complicate tissue preservation and necessitate decalcification. Cryosection immunostaining offers several advantages over alternative preparations (such as whole-mount immunostaining). The key benefits of cryosectioning are the general preservation of proteins and nucleic acids, and the relatively thin, ~2D sections allow for consistent and precise measurements. In addition, it allows all cochlear cell types to be preserved and visualized, and it can improve immunostaining quality. Unlike many cochlear whole-mount preparations, cryosectioning preserves structures including the spiral ligament, stria vascularis, Reissner's membrane, and tectorial membrane. See the following prior publications for examples of high-quality section immunostaining of embryonic^19,20,21, neonatal^22,23,24, and adult^25,26 inner ears.

NOTE: All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Georgetown University. All mice in this study were maintained in accordance with the Georgetown University Institutional Animal Care and Use Committee (protocol #1147). In the first postnatal week, because the temporal bone structures are not fully developed, decalcification is not required. However, cochleae from mice aged P6 and older require decalcification and a different method of dissection. In our protocol, male and female NCI Cr:NIH(S) (NIH Swiss) mice were used, and at E16.5, P0, and P30. For each section, we note in parentheses how long each step is expected to take for beginners. Many of the steps will become faster with practice.

1. Embryonic and juvenile inner ear sample collection (~75 min)

1. Euthanize and decapitate mice in accordance to an approved animal study protocol.
2. Using fine dissection scissors, carefully make a midline incision along the scalp, starting from the neck and extending toward the snout, pushing the skin sideways (first cut).
3. Place the bottom scissor blade at the foramen magnum and the top scissor blade at the cranium. Cut the top half of the cranium up to the nose (second cut).
4. Back the scissors out of the partially incised head, then place the bottom scissor blade at the bottom of the cranium and the top scissors end at the foramen magnum. Cut the bottom half of the head up to the nose (third cut).
   NOTE: It is important that the scissor blades cut right through the midline and cut easily with little effort. This will ensure that the scissor blades cut right in between the left and right inner ear.
5. At this point, the mouse head is divided in half along the rostral-caudal axis. Using fine forceps and scissors, carefully detach any soft tissue surrounding the temporal bone, such as brain, muscles, and connective tissue. Avoid damaging the bone or surrounding structures.
6. Place the mouse half-heads in wells of a 24-well plate containing room temperature 1x PBS. Once each head has been processed, exchange the 1x PBS solution with 4% PFA solution (for fixation) and incubate for 45 min at room temperature (typically 20 °C).
   NOTE: The fixation time may vary depending on the proteins that need to be detected and/or the binding efficacy of different antibodies.
7. After 45 min, rinse the tissue for 3 x 5-10 min with 1x PBS.
8. Store the samples in 1x PBS at 4 °C or go to the next step and dissect them. For long-term storage, store in 1x PBS containing 0.1% sodium azide at 4 °C.
9. Dispose of any remaining mouse tissue according to the institution's guidelines for biohazard/tissue disposal.

2. Embryonic and juvenile inner ear sample dissection (3 - 5 min each sample)

1. Locate the temporal bone/inner ear capsule within the mouse half-head. It occupies the same position as the outer ear but is situated inside the skull bone. Using fine forceps, carefully detach any soft tissue surrounding the inner ear capsule. Once the inner ear capsule is loosened, start cutting the tissue around the inner ear capsule.
2. Once the inner ear capsule has been mostly freed from the skull tissue, gently apply pressure to the surrounding area of the inner ear capsule using forceps to free it completely.
   NOTE: Apply gradual pressure to loosen the inner ear capsule from its surrounding attachments. Avoid damaging the inner ear capsule. Because the early developing inner ear is quite soft, avoid making direct contact with your forceps.
3. Store the inner ear samples as in step 1.8 and discard any remaining debris from the fixed head tissue.

3. Adult inner ear sample collection and dissection (~75 min + 2 - 3 days of EDTA treatment)

1. Euthanize and decapitate mice according to an approved animal study protocol.
2. Using sharp dissection scissors, carefully make a midline incision along the scalp, starting from the neck and extending toward the snout. Fold back the skin to expose the skull (first cut).
3. Place the bottom scissor blade at the foramen magnum and the top scissor blade at the cranium. Cut the top half of the cranium up to the nose (second cut).
   NOTE: During this step, you will notice moderate "crunching" of the scissor blades; this is normal.
4. Back the scissors out of the partially incised head, then place the bottom scissor blade at the bottom of the cranium and the top scissor blade at the foramen magnum. Cut the bottom half of the head up to the nose (third cut), being careful to go right along the midline to ensure the blades miss the inner ears.
   NOTE: Do not force the scissors through the tissue; this could indicate the blades hitting one of the inner ears.
5. For each half-head, using forceps and scissors, detach any soft tissue surrounding the temporal bone, such as the brain, muscles, and connective tissue.
   NOTE: Take caution to avoid damaging the temporal bone or surrounding structures.
6. With the surrounding tissue cleared, "reverse curl" the skull tissue with fingers and use a thumb to gently apply pressure to the underside of the temporal bone. Apply gradual pressure while loosening the bone from its surrounding attachments.
   NOTE: As pressure is applied, the inner ear capsule gradually separates from the surrounding bones of the skull. With gentle manipulation, the bone should pop out relatively easily.
7. Once the inner ear capsule is removed, rinse it with 1x PBS.
8. Fill a sylgard dish with 4% PFA and place it under the microscope. Place a single popped-out inner ear in the sylgard dish.
9. Examine the inner ear for any attached soft tissue or bone and gently remove any.
10. Find the oval window, and gently remove the stapes with forceps.
11. Find the round window and poke a little hole to make sure no surrounding tissue is clogging it.
12. Open a small hole in the apex with fine forceps. Flush the cochlea with a P20 pipette filled with 4% PFA to fix. Be sure to visualize fluid (dilute blood, endolymph/perilymph) coming out of the oval window.
13. Place the fixed cochlea in a 24-well plate filled with 4% PFA. Mark the time. Incubate 30-60 min at room temperature with gentle agitation (rocker or nutator), then rinse the sample for 3 x 5-10 min with 1x PBS solution.
14. After the final rinse, place the tissue in 1.25 mM EDTA and allow it to rock for 2-3 days at 4 °C.
15. Dispose of the remaining mouse tissue appropriately according to the institution's guidelines for biohazard/tissue disposal.
16. After the EDTA treatment, rinse the tissue for 3 x 5 min with 1x PBS.
17. Store the samples in 1x PBS at 4 °C or go to the next step and dissect them. For long-term storage, store the samples in 1x PBS containing 0.1% sodium azide at 4 °C.

4. Inner ear sample preparation for cryosectioning (~24 h)

1. Place the dissected inner ear capsule in 10% sucrose (in 1x PBS) and let it incubate at room temperature for a minimum of 2 h.
   NOTE: When the samples have equilibrated fully, they will sink to the bottom of the tube.
2. Discard the 10% sucrose solution, add 20% sucrose in 1x PBS, and allow an additional 2 h of incubation at room temperature.
3. Discard the 20% sucrose solution, add 30% sucrose in 1x PBS, and incubate at 4 °C overnight (minimum).
   NOTE: This step can be reduced to 2 h, but overnight incubation yields the best results. The tissue can be left in any percentage of sucrose for an extended amount of time.
   1. Take out half of the 30% sucrose solution and fill the space with Optimal Cutting Temperature (OCT) compound. Let it rock for a minimum of 30 min and a maximum of 2 h.
      NOTE: Leaving the samples in OCT for an extended time will cause tissue to shrink.
4. Transfer the samples to labeled cryomolds that are filled with OCT.
5. Check the cochlear orientation under the microscope in the embedded cryoblock. For "standard" cochlear cross-sections, confirm that the concave side of the inner ear is facing the narrow sides of the cryomold.
6. Place dry ice in a small container and add 5-10 mL of ethanol or dimethylbutane. Place the cryomolds + OCT + sample on bubbling dry ice to rapidly freeze the block.
7. Store the samples at -80 °C.

5. Sectioning inner ears (~25 min per sample)

1. Transfer the cryoblocks to the chamber of a cryostat prechilled to -20 °C. Allow the samples to equilibrate there for 30-60 min.
2. Using a pencil, mark the side of the cryoblock that corresponds to the cochlear position to ensure proper orientation for sectioning.
3. Add a small pool of OCT to the chuck and place the cryoblock on it, broad side (without the pencil mark) down.
4. Place the chuck + OCT + sample in the chilled cryostat chamber, allowing the OCT to freeze completely. After a few minutes, place the chuck + sample on the chuck holder with the pencil mark pointing to the right or left.
5. Trim the block until the tissue is apparent (use 40 μm as the trim setting).
6. Once the tissue is apparent, harvest a 12 μm section with slides designed for cryosectioning and check the sectioning location under a microscope.
7. If approaching cochlear turns, harvest 12 μm sections, checking the position occasionally, until past all the turns.
   NOTE: The numbers of sections per slide can vary, but we typically get 8-10 sections per slide, and around 6 slides per cochlea.
8. Store the slides at -80 °C.

6. Section immunostaining (~1 - 3 days)

1. Remove the slides from the -80 °C freezer and allow them to air dry for approximately 10 min.
2. Using a pencil, label the slides with the antibodies that will be used.
3. Trace the edges using a hydrophobic marker (e.g., Hydrophobic Barrier Peroxidase-Antiperoxidase Pen (PAP)).
4. Rehydrate the samples in 1x PBS for 5 min.
5. Discard the 1x PBS from slides and add 1x PBS + 0.1% Triton X-100 (0.1% PBSTx) to the slides for 20 min to permeabilize.
6. Remove 0.1% PBSTx from the slide and add 200 µL of the blocking solution for a 1-2 h incubation at room temperature.
   NOTE: Blocking is a crucial step in immunostaining techniques like immunohistochemistry and immunofluorescence, where it prevents the non-specific binding of antibodies to tissues or cells. For further information, see Lewis' guide to selecting control and blocking reagents²⁷. A commonly used blocking solution is 10% Normal Donkey Serum in 0.5% PBSTx, but this only applies when secondary antibodies derived from donkey are used.
7. Do a quick rinse with 1x PBS, add 200 µL of primary antibody solution made up in 0.5% PBSTx, and incubate for 1 h at room temperature or overnight at 4 °C. For the primary antibodies shown in Figure 1, use the following dilutions: 1:200 for Plexin-B1, 1:200 for Sox2, 1:1,000 each for Tuj1, Myo6 and Myo7A (see also Materials List).
   NOTE: Every primary antibody behaves differently depending on its epitope binding characteristics and ability to penetrate tissue samples.
8. Following the treatment with primary antibodies, rinse for 4 x 30 min in PBSTx, add 200 µL of secondary antibody solution made up in 0.5% PBSTx (Materials List), and incubate for 1 hour at room temperature.
   NOTE: Sometimes it can be helpful to spin secondary antibody solutions at full speed at 4 °C for 30 min to pellet any unwanted precipitate. If the secondary solutions have been centrifuged, avoid touching the bottom of the tube with the pipette tip.
9. Rinse for 4 x 30 min in PBSTx (or rinse overnight), then mount the slides using a mounting medium that helps prevent photobleaching (e.g., Fluoromount).
10. Add an appropriately sized coverslip with a thickness that is appropriate for the imaging setup (e.g., no. 1 coverslips, which are 0.13-0.16 mm in thickness).

We present examples from our own laboratory of the apex and basal turn of an embryonic day 16.5 (E16.5) mouse cochlea and the basal turn from postnatal day 0 (P0) and P30 cochleae. These samples have been sectioned and immunostained using markers for hair cells (Myo7A and Myo6), spiral ganglion neurons (SGNs; Tuj1), glia and support cells (Sox2), and Plexin-B1, which labels the basement membrane around the cochlear duct and SGNs. The cross-sections of the cochlea at different developmental stages reveal significant diffe...

There are several key steps for successful cryosectioning and immunostaining. Proper fixation of the cochlear tissue is essential, and the duration of fixation is also important, which can vary depending on the antibody. While most antibodies work better with a shorter fixation time, such as 45 min in PFA, some target epitopes can tolerate overnight fixation. Based on our experience, a 45 min fixation ensures adequate preservation and is compatible with most primary antibodies commonly used for research using cochlear pr...

The authors have no conflicts of interest to disclose.

M.A.D. and T.M.C. were supported by NIH grants 5R01DC016595-07 (to T.M.C.) and 5R01DC018040-05 (Michael Deans, Univ. Utah, PI). Example micrographs in Figure 1A-H were generated by Dr. Kaidi Zhang.