Cochlear Organ Dissection, Immunostaining, and Confocal Imaging in Mice

Materials and reagents

Biological materials

1. Rab11a conditional knock-out alleles, Vangl2-Looptail mice (The Jackson Laboratory, catalog number: 000220)

Reagents

1. Phosphate-buffered saline (PBS) (GENOM, catalog number: GNM20012-5)

2. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127)

3. Triton X-100 (Sigma, catalog number: 9002-93-1)

4. Normal donkey serum (Merck, Millipore, catalog number: S30)

5. Primary antibodies:

a. Rab11a, 1:200 (Cell Signaling Technology, catalog number: 2413)

b. γ-tubulin, 1:200 (Sigma, catalog number: T6557)

c. Arl13b, 1:1500 (Tamara Caspary, Emory University, Atlanta, GA)

d. Vangl2, 1:200 (R&D Systems, catalog number: AF4815)

e. Fz3, 1:500 (gift from Jeremy Nathans, Johns Hopkins University, Baltimore, MD)

f. LGN, 1:200 (gift from Fumio Matsuzaki, RIKEN)

g. β-Spectrin, 1:200 (BD Transduction Laboratories, catalog number: 612562)

h. MyosinVIIa, 1:200 (Proteus Bioscience Inc, catalog number: 25-6790)

i. Radixin, 1:100 (Abcam, catalog number: ab52495)

j. E-Cadherin, 1:200 (Invitrogen, catalog number: 13-1700)

6. Secondary antibodies:

a. Alexa Fluor^® 488 AffiniPure donkey anti-mouse IgG (H+L), 1:1,000 (Jackson Immuno-Research Laboratories, catalog number: 715-545-151)

b. Rhodamine Red^TM-X AffiniPure donkey anti-mouse IgG (H+L), 1:1,000 (Jackson Immuno-Research Laboratories, catalog number:715-295-151)

c. Alexa Fluor^® 647 AffiniPure donkey anti-mouse IgG (H+L), 1:1,000 (Jackson Immuno-Research Laboratories, catalog number:715-605-151)

7. Phalloidin, 1:1,000 (Sigma-Aldrich, catalog number: P5282)

8. Mounting medium (Vectashield Antifade Medium, catalog number: H-1200)

9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014)

10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)

11. Sodium phosphate dibasic heptahydrate (NaH₂PO₄·7H₂O) (Sigma-Aldrich, catalog number: S9390)

12. Potassium dihydrogen phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791)

Solutions

1. 1× PBS (see Recipes)

2. 4% PFA (see Recipes)

Recipes

1. 1× PBS

Adjust pH to 7.4 with HCl and store it at room temperature.

2. 4% PFA

Store paraformaldehyde powder at 4 °C. Heat while stirring under the chemical hood at approximately 60 °C. When the solution is transparent, let it cool down and then filter with Whatman paper to remove undissolved particles. Freeze PFA aliquots in 50 mL tubes at -20 °C. Remember to wear suitable personal protective equipment.

Equipment

1. Dissection microscope (BT, catalog number: BTS-300)

2. Large forceps (Fine Science Tools, catalog number: 11026-15)

3. Fine forceps (Fine Science Tools, catalog number: 11252-00)

4. Very fine forceps (Fine Science Tools, catalog number: 11200-14)

5. Small scissors (Fine Science Tools, catalog number: 91460-11)

6. Confocal microscope (Carl Zeiss Microscopy, model: LSM 510, AxioObserver)

7. 48-well plate (Corning, catalog number: 3548)

8. Petri dish (BD Biosciences, Falcon^®, catalog number: 351006)

Software and datasets

1. ImageJ software (http://rsb.info.nih.gov/ij)

Procedure

Below, we describe the step-by-step procedure for cochlear organ dissection, immunostaining, and confocal imaging in mice from E18.5 to P2.

A. Cochlear organ dissection

1. Euthanize the mouse via cervical dislocation or CO₂ inhalation (100% CO₂).

2. Decapitate the mouse with small scissors. Using large forceps, remove the brain on each side to identify the temporal bones.

3. Under a dissecting microscope, use fine forceps to isolate the bony labyrinths from the temporal bones.

4. Trim away additional tissue and remove the stapes from the oval window.

5. Use the tip of the fine forceps to clear the oval and round windows and make a small hole in the apex of the cochlear spiral. Fix the bony labyrinths in 1.5 mL of 4% PFA for 5 min on ice.

6. Place the bony labyrinths in a dissecting dish containing PBS to facilitate the next step (Figure 2A).

Figure 2. Cochlear organ dissection. A) After the removal of additional tissue from the temporal bone, the otic vesicles are revealed under a light microscope. B) Dissected organ of Corti.

7. Use fine forceps to remove the outer cartilage to expose the cochlear duct.

8. Use fine forceps to slowly remove the stria vascularis. Remove the whole stria in one attempt as far as possible.

9. Use very fine forceps to remove the Reissner’s membrane. Visualize the dorsal aspect of the cochlear duct, including the sensory epithelium.

10. Use very fine forceps to remove the tectorial membrane.

11. Use very fine forceps to remove the intact organ of Corti (Figure 2B, Video 1).

12. Fix the organ of Corti immediately in the 48-well plate containing 0.5 mL of 4% PFA for 2 h at room temperature (RT), 2 h on ice, or overnight at 4 °C.

B. Immunostaining

1. Transfer the organ of Corti to a different well in the 48-well plate containing 0.5 mL of 0.1% Triton X-100 in PBS (PBS-T) for 10–20 min at RT. Agitation is not required for any of these steps. Transfer the organ of Corti with very fine forceps.

2. Place the tissues in 10% normal donkey serum and PBS-T for 1 h at RT.

3. Incubate the organ of Corti tissues in primary antibody in 5% normal donkey serum and PBS-T overnight at 4 °C.

4. Wash with 0.5 mL of PBS-T three times, 1–2 h each, at RT.

5. Incubate the samples in secondary antibodies in 5% normal donkey serum and PBS-T for 2 h at RT or overnight at 4 °C. This procedure should be shielded from light.

6. Wash the sample with PBS-T three times every 1–2 h at RT.

7. Incubate the tissue in phalloidin in 5% normal donkey serum and PBS-T for 30 min at RT.

8. Wash the sample with PBS-T three times every 1–2 h at RT.

C. Confocal imaging

1. Gently transfer the basilar membrane into a drop of PBS placed on a microscope slide.

2. Under the microscope, use very fine forceps to orient the sample facing upward toward the coverslip.

3. Add a drop of mounting medium directly onto the basilar membrane sample. Place one side of the coverslip at an angle against the slide, making contact with the outer edge of the liquid drop, and then lower the cover gently. Take care to avoid air bubbles.

4. Use an adsorbent tissue or a piece of filter paper to remove any excess mounting medium from the edges of the coverslip.

5. Seal the coverslip to the glass slide with nail polish. The sample should be stored at 4 °C in a dark environment until observation.

6. Check the intensity of the labeling and evaluate the background of the experiment by looking at the negative controls.

7. Acquire all pictures in the same conditions (exposure and general settings) for each color channel.

8. Visualize with Zeiss LSM510 confocal microscope equipped with the ZEN software using 20–63× objectives (oil immersion, NA 1.4) and the 488- and 555-nm excitation wavelengths of the laser.

9. Use the Z projection function to provide a two-dimensional view of all the pictures of an image stack by projecting them along the axis perpendicular to the image plane. The Maximum Intensity option creates an image in which each pixel contains the maximum value over all images in the stack.

10. Define specific positions along the cochlear duct, such as 25%, 50%, and 75% from the base, and use these to compare the proteins of interest in cochlear regions.

11. Take pictures using the microscope software for quantification of the particular phenotype as required.

a. Stereocilia morphology: Measure the bundle convexity or the height, defined as the shortest distance between the vertex of the bundle and the line that connects two ends of the bundle. For each genotype, three independent cochleae were counted and quantified, noting the region and hair cell type.

b. Stereocilia orientation: Assess the orientation of each individual bundle relative to a line perpendicular to the planar axis. The angle is defined between the center line and the horizontal line. One line is drawn to bisect the middle of the V-shaped stereocilia bundle, and another line is drawn perpendicular to the planar axis.

c. Cilia quantification: Cilia marked by Arl13b immunostaining are recorded and counted according to different IHC rows and OHC rows. A minimum of 300 IHCs and 1,200 OHCs are counted per region of each genotype. Five individual animal samples are included in the quantification.

12. The above procedures are completed using ImageJ software. The specific steps of the software are as follows (Figure 3):

a. Open the images in ImageJ.

b. Use the straight line tool from the toolbar to connect two ends of the bundle.

c. Use the straight line tool from the toolbar to measure the shortest distance between the vertex of the bundle and the line.

d. Click Analyze > Measure and the length value will appear.

e. Use the angle tool from the toolbar to measure the V-shaped stereocilia bundle.

f. Use the straight line tool from the toolbar to bisect the middle of the V-shaped stereocilia bundle (angle 1 and angle 2 are equal).

g. Use the angle tool from the toolbar to measure the angle between the bisect line and the horizontal line (angle 3).

h. Click Analyze > Measure and the angle value will appear.

Figure 3. Cochlear hair cells and quantification of the particular aspects. A) Phalloidin staining (green) in the cochlea, showing the stereocilia bundles of hair cells. B) Zoomed-in view of a single hair cell. C) Schematic diagram of length measurement. The red line indicates the length. D) Schematic diagram of angle measurement. One line indicates the bisector of the angle in V-shaped stereocilia bundles (angle 1 and angle 2 are equal). The other line indicates the horizontal line (angle 3, marked in red, indicates the measurement angle).

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

1. Knapp et al. [14]. Rab11a Is Essential for the Development and Integrity of the Stereocilia and Kinocilia in the Mammalian Organ of Corti. eNeuro (Figure 1, panels A–D; Figure 4, panels A–E; Figure 5, panels A–H; Figure 6, panels A and B; Figure 7, panels F–I).

General notes and troubleshooting

Troubleshooting

Problem: Unsatisfactory immunostaining results.

Possible cause: Poor quality of samples collected; poor quality of antibodies used; irregular staining procedures.

Solution: Fresh samples should be used as much as possible to prevent problems such as degradation of proteins and severe dehydration. Ensure that the antibody is within its expiration date and use the same batch of antibody as much as possible. Note that it is necessary to avoid light when staining the secondary antibody.

Acknowledgments

Funding was provided by the National Natural Science Foundation of China (NSFC, Grants 82271166, 81970880, and 81771017 to D.R., 82201288 to X.Q.) and by the Natural Science Foundation of Shanghai (Grant No. 22ZR1410100 to D.R.). We thank eNeuro Journal for some illustrating images we used in this paper [14].

Competing interests

The authors declare no competing interests.

Ethical considerations

All procedures were approved by the Emory University Institutional Animal Care and Use committee and met the NIH guidelines.

References

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