Name: whole mount dissection and immunofluorescence of the adult mouse cochlea
Uploaded: 2016-01-01T15:00:00
Description: Watch this Scientific Journal Video about Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea at JoVE.com

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).

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
<ol>
	<li>Identify temporal bones in the base of the mouse skull13 and scrape away the cranial&#160;nerves using standard pattern forceps.</li>
	<li>Place standard pattern forceps at the tip of the otic capsule and with the thumb of the opposite hand press down on the posteri...

<ol>
	<li>Robles, L., Ruggero, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+the+mammalian+cochlea.">Mechanics of the mammalian cochlea.</a> Physiol Rev. 81 (3), 1305-1352 (2001).</li><li>Ehret, G., Frankenreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitattive+analysis+of+cochlear+structures+in+the+house+mouse+in+relation+to+mechanisms+of+acoustical+information+processing.">Quantitattive analysis of cochlear structures in the house mouse in relation to mechanisms of acoustical information processing.</a> J Comp Physiol. 122, 65-85 (1977).</li><li>Ou, H. C., Bohne, B. A., Harding, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+damage+in+the+C57BL/CBA+mouse+cochlea.">Noise damage in the C57BL/CBA mouse cochlea.</a> Hear Res. 145 (1-2), 111-122 (2000).</li><li>Engstrom, H., Ades, H. W., Andersson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structural pattern of the organ of Corti. A systematic mapping of sensory cells and neural elements. , (1966).</li><li>Grant, L., Yi, E., Goutman, J. D., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+Recordings+at+Afferent+Dendrites+Contacting+Cochlear+Inner+Hair+Cells:+Monitoring+Multivesicular+Release+at+a+Ribbon+Synapse.">Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse.</a> J Vis Exp. (48), e2442 (2011).</li><li>Bohne, B. A., Harding, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+and+analyzing+the+mouse+temporal+bone+to+identify+gross,+cellular+and+subcellular+pathology.">Processing and analyzing the mouse temporal bone to identify gross, cellular and subcellular pathology.</a> Hear Res. 109 (1-2), 34-45 (1997).</li><li>Bohne, B. A., Harding, G. W., Ou, H. C., Willott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Mouse Auditory Research. , 171-187 (2001).</li><li>Weber, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+cell-cycle+reentry+and+cell+death+after+acute+inactivation+of+the+retinoblastoma+gene+product+in+postnatal+cochlear+hair+cells.">Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells.</a> Proc Natl Acad Sci U S A. 105 (2), 781-785 (2008).</li><li>Yu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+proliferation+of+postmitotic+cochlear+supporting+cells+by+acute+ablation+of+the+retinoblastoma+protein+in+neonatal+mice.">In vivo proliferation of postmitotic cochlear supporting cells by acute ablation of the retinoblastoma protein in neonatal mice.</a> J Neurosci. 30 (17), 5927-5936 (2010).</li><li>Steigelman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystin-1+Is+Required+for+Stereocilia+Structure+But+Not+for+Mechanotransduction+in+Inner+Ear+Hair+Cells.">Polycystin-1 Is Required for Stereocilia Structure But Not for Mechanotransduction in Inner Ear Hair Cells.</a> J Neurosci. 31 (34), 12241-12250 (2011).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+in+vivo+conversion+of+mouse+cochlear+pillar+and+Deiters'+cells+to+immature+hair+cells+by+Atoh1+ectopic+expression.">Age-dependent in vivo conversion of mouse cochlear pillar and Deiters' cells to immature hair cells by Atoh1 ectopic expression.</a> J Neurosci. 32 (19), 6600-6610 (2012).</li><li>Parker, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> J Vis Exp. (36), e1685 (2010).</li><li>Burns, J. C., On, D., Baker, W., Collado, M. S., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+half+the+hair+cells+in+the+mouse+utricle+first+appear+after+birth,+with+significant+numbers+originating+from+early+postnatal+mitotic+production+in+peripheral+and+striolar+growth+zones.">Over half the hair cells in the mouse utricle first appear after birth, with significant numbers originating from early postnatal mitotic production in peripheral and striolar growth zones.</a> J Assoc Res Otolaryngol. 13 (5), 609-627 (2012).</li><li>Collado, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+postnatal+accumulation+of+junctional+E-cadherin+is+inversely+correlated+with+the+capacity+for+supporting+cells+to+convert+directly+into+sensory+hair+cells+in+mammalian+balance+organs.">The postnatal accumulation of junctional E-cadherin is inversely correlated with the capacity for supporting cells to convert directly into sensory hair cells in mammalian balance organs.</a> J Neurosci. 31 (33), 11855-11866 (2011).</li><li>Meyers, J. R., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+change+controls+supporting+cell+proliferation+in+lesioned+mammalian+balance+epithelium.">Shape change controls supporting cell proliferation in lesioned mammalian balance epithelium.</a> J Neurosci. 27 (16), 4313-4325 (2007).</li><li>Gratton, M. A., Rao, V. H., Meehan, D. T., Askew, C., Cosgrove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+dysregulation+in+the+stria+vascularis+of+mice+with+Alport+syndrome:+implications+for+capillary+basement+membrane+pathology.">Matrix metalloproteinase dysregulation in the stria vascularis of mice with Alport syndrome: implications for capillary basement membrane pathology.</a> Am J Pathol. 166 (5), 1465-1474 (2005).</li></ol>

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 fixat...

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&#39;s lab at St. Jude Children&#39;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.

<table border="0" cellpadding="0" cellspacing="0" width="1074">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:377px;">Standard&#160; pattern forceps</td>
			<td style="width:139px;">Fine Science Tools</td>
			<td style="width:163px;">11000-12</td>
			<td style="width:395px;">can be purchased from other vendors</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">10.5 cm fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml microcentrifuge tubes</td>
			<td>MidSci</td>
			<td>AVSS2000</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">16% formaldehyde, methanol free, ultra pure, EM grade</td>
			<td>Polysciences</td>
			<td>18814</td>
			<td style="width:395px;">TOXIC --wear gloves and cannot be disposed of in the sink. Can be purchased from other vendors.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS pH 7.4&#160;</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher</td>
			<td>BP118-500</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">end-over-end tube rotator</td>
			<td>Fisher</td>
			<td>05-450-127</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm petri dish</td>
			<td>Fisher</td>
			<td>50-202-037</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dow Corning Sylgard 184 silicone encapsulant kit</td>
			<td>Ellsworth Adhesives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">activated charcoal</td>
			<td>Fisher</td>
			<td>AC134372500</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stereo dissection microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #4 jeweler&#39;s forceps</td>
			<td>Fine Science Tools</td>
			<td>11241-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 jeweler&#39;s forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5 mm Vannas spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td>curved&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mm Vannas-Tubingen spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td>straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48 well plates</td>
			<td>Fisher</td>
			<td>08-772-52</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8 well chamber slides</td>
			<td>Fisher</td>
			<td>1256518</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA, fraction V</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGS</td>
			<td>Vector labs</td>
			<td>S-1000</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NHS</td>
			<td>Vector labs</td>
			<td>S-2000</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D rotator</td>
			<td>Midsci</td>
			<td>R3D-710</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Western blot incubation box XL</td>
			<td>Licor</td>
			<td>929-97401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Prolong gold antifade mounting media</td>
			<td>Life Technologies</td>
			<td>P36930</td>
			<td style="width:395px;">can be purchased from other vendors,but mounting medias vary in their ability to protect against photobleaching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips 22 x 22 x 1</td>
			<td>Fisher</td>
			<td>12-548-B</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">clear nail polish</td>
			<td>Local drug store</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cardboard slide folder</td>
			<td>Fisher</td>
			<td>12-587-10</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic slide box</td>
			<td>Fisher</td>
			<td>03-448-10</td>
			<td>can be purchased from other vendors</td>
		</tr>
	</tbody>
</table>

whole mount dissection and immunofluorescence of the adult mouse 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.

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...

Watch this Scientific Journal Video about Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea at JoVE.com

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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 ...

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