We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.
The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.
The spiral-shaped cochlea of the inner ear, contained within the temporal bone, houses the organ of Corti, the auditory sensory end organ in mammals. The cochlea is tonotopically organized and commonly divided into apical, middle, and basal turns corresponding to different frequency regions with high frequency sound detection in the base and low frequency detection in the apex1. Hair cells, the mechanosensory cells of the organ of Corti, run the length of the cochlea, which is approximately 6 mm long in mice2,3. These cells convert the mechanical energy of sound waves, which are transmitted through the fluid-filled membranous labyrinth, into neural signals that are processed by central auditory structures. The technique described here provides a method for preparing whole mounts of the organ of Corti after calcification of the cochlea is complete (for samples ranging from one week of age to adulthood). We also present a method for immunostaining the whole mounted cochlear tissue. Cochlear whole mounts are crucial for visualization of all hair cells and surrounding supporting cells in their natural spatial arrangements and allow for analysis in three dimensions with the use of confocal microscopy.
Drs. Hans Engstrom and Harlow Ades originally described a whole mount cochlear dissection method in 1966. They detailed a technique to rapidly fix and dissect calcified cochleae submerged in liquid from a variety of mammals, preserving short intact segments of the organ of Corti for microscopic analysis4. The dissection of an unfixed, calcified rat cochlea has also been illustrated in an instructional video5. Drs. Barbara Bohne and Gary Harding at Washington University made several important modifications to this method. In their version of the cochlear whole mount method, the temporal bone was decalcified, embedded in plastic, and five half-turns or ten quarter-turns were dissected6,7. Dr. Charles Liberman and colleagues at Eaton Peabody Laboratories, Massachusetts Eye and Ear Infirmary, modified this technique so that plastic embedding was not required8. Further modification of the technique occurred in Dr. Jian Zuo's lab at St. Jude Children's Research Hospital9-12 which informed the dissection method presented here. We use a different strategy to gain access to the organ of Corti than Bohne and Liberman, which allows isolation of complete apical, middle, and basal turns. Thus the dissected tissue is larger and less likely to be lost or damaged during the dissection or immunostaining processes. In addition, the current method facilitates measurement of the distance from the apical tip or basal hook to identify a frequency region.
Although many labs perform immunostaining of cochlear tissue, it is unclear where this method originated. As a result there are various recipes for blocking buffers and antibody incubation buffers that may affect the performance of individual primary antibodies. Here, we present one method for immunostaining with fluorescently tagged antibodies that is applicable to most commonly used antibodies in the auditory field.
The complex shape of the cochlea, delicate structure of the organ of Corti, and bony encasement provide a challenge for histological and biochemical analysis. A variety of techniques are currently used in the hearing field to surmount these difficult features and visualize the cells within the organ of Corti, each technique with its own advantages and disadvantages. The protocol presented here allows for whole mount dissection of the adult mouse cochlea and, with slight modification, can potentially be used to examine the critical structures within the cochleae from a variety of other model organisms used in the field.
Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Southern Illinois University School of Medicine.
1. Extraction of Temporal Bones
2. Post-fix Temporal Bones
3. Decalcify Temporal Bones
4. Create Silicone Elastomer-coated Dissection Dish
5. Whole Mount Dissection of the Cochlea (for P7 and Older Samples)
6. Immunostaining with Fluorescently Tagged Antibodies
7. Mount Cochlear Turns on Slides
We present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base) from cochlear tissue that is calcified, with key dissection steps presented in Figure 1. During the first postnatal week of development, calcification of the mouse cochlea is incomplete and a more simple dissection method can be used13. Using the neonatal whole mount dissection method with cochlea from P7 and older mice results in tears and shredding of the organ of Corti. The spiral ligament/lateral wall is now more firmly attached and cannot be peeled away from the sensory epithelium without causing damage. Thus the "adult" whole mount dissection method is needed for samples older than P6. We present an example of the middle turn of a P15 mouse cochlea that has been dissected and immunostained with hair cell and supporting cell markers (Figure 2). Optical cross-sections can also be obtained with the whole mount technique (Figure 3).
Several problems can occur during the whole mount dissection or when mounting the cochlear turns on slides. During the removal of the spiral ligament/lateral wall, there is a narrow window between cutting too much or not enough. Cuts that occur next to the last row of outer hair cells may cause the hair cells in this last row to mount at varied angles (Figure 4A). Cuts that are too large can remove sections of the organ of Corti (Figure 4B). Handling the sample with forceps takes great care and often there are holes in the organ of Corti where forceps were misplaced (Figure 4C). Finally when mounting the cochlear turns, the organ of Corti can fold which obscures the image (Figure 4D).
Figure 1. Important Steps in the Whole Mount Dissection of the “Adult” Mouse Cochlea. (A) After protocol step 5.1.4, the basal turn of the cochlea is separated from middle/apical turns, yet still attached to the vestibular region. (B) After protocol step 5.3.3, the middle turn is separated from the apical turn. (C) Example of the completed dissection of a middle turn where the spiral ligament/lateral wall is removed. Please click here to view a larger version of this figure.
Figure 2. Confocal Slice Image of the Middle Turn Isolated from a P15 Mouse. Four 20x images are overlaid to reconstruct the whole middle turn. Hair cells are labeled with a rabbit anti-myosin VIIa primary antibody (1:200 dilution) combined with a donkey anti-rabbit Alexa 488-conjugated secondary antibody (1:1,000 dilution) (magenta). Supporting cells are labeled with a goat anti-Sox2 primary antibody (1:500 dilution) combined with a donkey anti-goat Alexa 568-conjugated secondary antibody (1:1,000 dilution) (green). Hoechst (blue) labels all nuclei. Image was taken using a Zeiss LSM 700 confocal microscope with 405, 488, and 555 wavelengths. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 3. Optical Cross-section of the Middle Turn Isolated from a 6-week Old Mouse. (A) Confocal slice image of the whole mount preparation (bottom) and optical cross-section in the XZ plane (top). (B) Increased magnification of the top panel in (A), with the crosshairs removed. Hair cells are labeled with a rabbit anti-myosin VIIa primary antibody (1:200 dilution) combined with a donkey anti-rabbit Alexa 647-conjugated secondary antibody (1:1,000 dilution) (magenta). Supporting cells are labeled with a goat anti-Sox2 primary antibody (1:500 dilution) combined with a donkey anti-goat Alexa 568-conjugated secondary antibody (1:1,000 dilution) (green). Image was taken using a Zeiss LSM 700 confocal microscope with 405, 555, and 647 wavelengths. Scale bars = 20 µm Please click here to view a larger version of this figure.
Figure 4. Examples of Problems that can Occur During the Whole Mount Dissection or When Mounting Cochlear Turns on Slides. (A) On the left side of the image, the cochlear tissue was cut next to the last row of outer hair cells causing many of these cells to be mounted at varied angles. (B) A section of the organ of Corti on the left side of the image has been cut off. (C) There is a hole punched in the outer hair cell region in the middle of the image. (D), The organ of Corti is folded in several places. Images were taken from 4 – 8 week old mouse cochleae. Hair cells are labeled with a rabbit anti-myosin VIIa primary antibody (1:200 dilution) combined with a goat anti-rabbit Alexa 488-conjugated secondary antibody (1:1,000 dilution) or a donkey anti-rabbit Alexa 488-conjugated secondary antibody (1:1,000 dilution) (magenta). In C outer hair cells are labeled with a goat anti-prestin primary antibody (1:200 dilution) combined with a donkey anti-rabbit Alexa 568-conjugated secondary antibody (1:1,000 dilution) (green). Images were taken using a Leica SP5 confocal microscope with 405, 488, and 555 nm wavelengths. Scale bars: in A-B = 20 µm; in C-D = 40 µm. Please click here to view a larger version of this figure.
There are several critical steps for successful whole mount dissection and immunostaining. However before either of these methods are performed, proper fixation of the cochlear tissue is needed. We recommend using methanol free, ultra-pure, EM grade PFA. PFA made from powder can have traces of methanol and an unstable pH which decreases the quality of immunofluorescence. Other groups have also shown that similar dissections are possible using fixatives that do not contain formaldehyde14-16. The length of fixation is also important and is antibody specific. Some antibodies can tolerate an O/N fixation, while others do not work well with just 1 hr in PFA (however this is rare). Under-fixed tissue can be problematic for the dissection method as the tissue falls apart. In our experience a 3 – 4 hr fixation provides adequate fixation and does not interfere with the majority of primary antibodies commonly used in the hearing field.
It is also possible that EDTA can interfere with primary antibodies; thus some antibodies will work well in neonatal tissue, but not in P7 or older tissue that was decalcified. After fixation, temporal bones can be stored for variable amounts of time before decalcification depending on the antigens being examined. Some antigens require decalcification and dissection within days to weeks after fixation, while others may be stored for years (either before or after decalcification) without decreasing the quality of the immunostaining. We recommend storing samples as temporal bones due to the risk of evaporation of the storage media (PBS) from the 48-well plate and potential contamination with fungus or bacteria. We typically perform the whole mount dissection less than one week in advance to immunostaining.
Once fixed and decalcified, the whole mount dissection is performed with the temporal bone submerged in liquid. Removing excess bone and soft tissue surrounding the labyrinth early in the dissection will aid in removal of the spiral ligament/lateral wall at later stages by facilitating the manipulation of the tissue and providing a less obscured view of critical structures. When performing the first few steps, forceps can be used to hold the tissue in the vestibular region. However once the turns are isolated, it is important to avoid placing forceps on the organ of Corti or spiral ligament/lateral wall. Instead, keep the forceps closed and pin the spiral ganglion nerve fibers to the silicone elastomer-coated dissection dish. Do not hold onto this region as the tissue will tear. In general, once the tissue has been divided into the three turns, grasping and pulling maneuvers can cause unpredictable results, which are often damaging to the organ of Corti and should be avoided. Tissue from younger animals (P7-P21) tends to be more forgiving than tissue from animals older than P21. In addition, cochlear samples with hair cell damage are more difficult to dissect. If the mouse received noise exposure the tissue is especially fragile. Regardless of the state of the tissue, the dissection we present is technically demanding and requires many practice attempts for proficiency.
During immunostaining, it is important that each cochlear turn is submerged in liquid, not floating on top or stuck to the side of the well. This allows more complete penetration of triton and antibodies into the tissue. When removing liquid from each well, it is easy to lose the cochlear turn or draw it up into the pipette tip. Changing solutions with a 200 µl pipette tip using a dissection scope will help prevent this. Slowly extract the liquid and move the pipette tip if the cochlear turn gets too close. Also pipetteting waste solution into a clean tube can be a good strategy as this waste tube can be searched if a turn is accidentally drawn up into the pipette. If the turn is stuck in the pipette tip, the tip can be cut open with a razor blade, but often the organ of Corti will be damaged if this occurs.
When trouble-shooting antibodies for immunostaining, additional steps such as antigen retrieval or signal enhancement can be added. There are low pH and high pH antigen unmasking reagents that can be purchased. If an antibody does not work with the method described here, the first protocol change to try is one of these antigen retrieval methods. Alternatively, use a signal enhancer. There are commercially available solutions to use prior to immunostaining, or tyramide amplification kits that can be used to amplify the signal from the secondary antibody.
The significance of the technique we present is the ability to maintain the three-dimensional structure of the organ of Corti and to visualize all cells within the organ. The entire length of the cochlea is separated into only three turns while other similar techniques, namely the Bohne and Liberman methods, require division into 5 – 10 pieces6-8, increasing the number of samples to maneuver in the immunostaining and imaging processes. The cochlear lateral wall dissection that was developed by Cosgrove and Gratton requires a similar level of skill and is possible in unfixed, fresh tissue, as well as in decalcified tissue, but the organ of Corti is stripped away and destroyed in the process of isolating the lateral wall17. Another group has performed similar dissections in unfixed, fresh cochlear tissue from three week old rats where the spiral ligament/lateral wall is grasped and stripped away from the organ of Corti, leaving the organ intact. However this was only achieved in the apical turn5. The method of peeling the spiral ligament/lateral wall away from the organ of Corti is routine for dissection of fixed tissue in a young mouse (< P7)13. However, in our experience with mice older than P6, after fixation and decalcification, this maneuver often tears the organ of Corti in an unreliable fashion. In addition the procedure described here allows isolation of middle and basal turns as well.
Cryosections and sections obtained after paraffin embedding are also commonly used in the auditory field. These methods allow visualization of other structures such as the stria vascularis, Reissner's membrane, and tectorial membrane, yet each section only allows visualization of a small region of the organ of Corti in each cochlear turn. Thus to investigate events that occur in a mosaic pattern, such as cell loss or cell division, with the sectioning method, 50 or more slides need to be stained and imaged to capture the entire length of the cochlea. In contrast, the whole mount dissection protocol has the advantage of preparing the entire organ of Corti in just three pieces. In addition to the benefit of preserving the lengthwise architecture of the organ of Corti, this technique allows for simplified data collection and storage. One limitation of the whole mount dissection is that it destroys surrounding structures such as the spiral ligament, stria vascularis, Reissner's membrane, and tectorial membrane. Another limitation is the technical difficulty and length of time for a single dissection. This is mostly due to the fragile nature of the organ of Corti and small margin for error when removing the spiral ligament/lateral wall. Once mastered, the whole mount dissection of one cochlea can be performed in about 20 – 30 min .
While the above protocol describes a whole mount dissection method for the adult mouse, we hope to apply this technique to other model organisms used in the auditory field. Our lab is currently modifying this technique for the dissection of the rat cochlea. The larger cochlea of the rat is encased in a thicker otic capsule that is more densely calcified than the mouse. Thus the temporal bone must be excised with large scissors. Before fixation and decalcification with EDTA, the otic capsule should be opened with scissors to allow better access to the cochlear tissue. Also the decalcification process is longer and can take up 3 weeks depending on the age of the sample. For the whole mount dissection, the size of the bony labyrinth affects the technique. The increased size of the rat cochlea provides a slightly larger distance between the spiral ligament/lateral wall and organ of Corti, providing a larger margin of error for individual cuts. However, while the larger tissue may provide more material to grasp for manipulation of the specimen, it also requires a greater number of cuts to remove the entire length of the spiral ligament/lateral wall. We believe that analogous modifications could be made for dissection of the cochlea from chinchilla, gerbil, and guinea pig.
The authors have nothing to disclose.
This work was supported in part by a grant from the Office of Naval Research (N000141310569). The Southern Illinois University School of Medicine Research Imaging Facility equipment was supported by the National Center for Research Resources-Health (S10RR027716).
Standard pattern forceps | Fine Science Tools | 11000-12 | can be purchased from other vendors |
10.5 cm fine scissors | Fine Science Tools | 14060-11 | can be purchased from other vendors |
2 ml microcentrifuge tubes | MidSci | AVSS2000 | can be purchased from other vendors |
16% formaldehyde, methanol free, ultra pure, EM grade | Polysciences | 18814 | TOXIC –wear gloves and cannot be disposed of in the sink. Can be purchased from other vendors. |
PBS pH 7.4 | Sigma | P3813-10PAK | can be purchased from other vendors |
EDTA | Fisher | BP118-500 | can be purchased from other vendors |
end-over-end tube rotator | Fisher | 05-450-127 | can be purchased from other vendors |
60 mm petri dish | Fisher | 50-202-037 | can be purchased from other vendors |
Dow Corning Sylgard 184 silicone encapsulant kit | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | |
activated charcoal | Fisher | AC134372500 | can be purchased from other vendors |
stereo dissection microscope | Zeiss | Stemi 2000 | can be purchased from other vendors |
Dumont #4 jeweler's forceps | Fine Science Tools | 11241-30 | |
Dumont #5 jeweler's forceps | Fine Science Tools | 11251-20 | |
2.5 mm Vannas spring scissors | Fine Science Tools | 15001-08 | curved |
5 mm Vannas-Tubingen spring scissors | Fine Science Tools | 15003-08 | straight |
48 well plates | Fisher | 08-772-52 | can be purchased from other vendors |
8 well chamber slides | Fisher | 1256518 | can be purchased from other vendors |
Triton X-100 | Sigma | X100-500 | can be purchased from other vendors |
BSA, fraction V | Fisher | BP1605 | can be purchased from other vendors |
NGS | Vector labs | S-1000 | can be purchased from other vendors |
NHS | Vector labs | S-2000 | can be purchased from other vendors |
3D rotator | Midsci | R3D-710 | can be purchased from other vendors |
Western blot incubation box XL | Licor | 929-97401 | |
Hoechst 33342 | Life Technologies | H3570 | can be purchased from other vendors |
Prolong gold antifade mounting media | Life Technologies | P36930 | can be purchased from other vendors,but mounting medias vary in their ability to protect against photobleaching |
Superfrost Plus Slides | Fisher | 12-550-15 | can be purchased from other vendors |
coverslips 22 x 22 x 1 | Fisher | 12-548-B | can be purchased from other vendors |
clear nail polish | Local drug store | can be purchased from other vendors | |
cardboard slide folder | Fisher | 12-587-10 | can be purchased from other vendors |
plastic slide box | Fisher | 03-448-10 | can be purchased from other vendors |