Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

Summary

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.

Abstract

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.

Introduction

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 mice^2,3. These cells convert the mechanical energy of sound waves, which are transmitted through the fluid-filled membranous labyrinth, into neur....

Protocol

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

1. Identify temporal bones in the base of the mouse skull¹³ and scrape away the cranial nerves using standard pattern forceps.
2. Place standard pattern forceps at the tip of the otic capsule and with the thumb of the opposite hand press down on the posterior semicircular canal to dislodge the encapsulated cochlea.
3. Free the bottom half of the temporal bone from the skull manually with the thumb....

Results

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.......

Discussion

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 formaldehyde^14-16. The length of fixat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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