Name: vibratome sectioning for enhanced preservation of the cytoarchitecture of the mammalian organ of corti
Uploaded: 2011-06-17T13:00:00
Description: Watch this Scientific Journal Video about Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti at JoVE.com

The author would like to acknowledge use of the Children's Research Institute Imaging Core for the confocal microscopy and Dr. Jian Zhang for the suggestion to mount sections using agarose beads. This work was funded by N.I.H. grant DC010387.

1. Isolation and fixation of inner ears
<ol>
 <li>For analysis of cellular patterning in the organ of Corti, euthanize appropriately staged embryos or adults.</li>
 <li>Dissect out the otic capsule, containing the membranous inner ear. In the mouse, this can be easily done on embryos at embryonic (E) day 14.5 and older, in which E0.5 is the day at which a vaginal plug was detected. Dissection of the otic capsule is accomplished first by decapitation and opening of the skull at the midline. Remove brain to expose eac...

<ol>
	<li>Lim, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+of+the+organ+of+Corti:+a+review.">Functional structure of the organ of Corti: a review.</a> Hear Res. 22, 117-146 (1986).</li><li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+innervation+of+the+cochlea.">Structure and innervation of the cochlea.</a> Brain Res Bull. 60, 397-422 (2003).</li><li>Chung, W. S., Shin, C. H., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp2+signaling+regulates+the+hepatic+versus+pancreatic+fate+decision.">Bmp2 signaling regulates the hepatic versus pancreatic fate decision.</a> Dev Cell. 15, 738-748 (2008).</li><li>Sun, X., Mariani, F. V., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+FGF+signalling+from+the+apical+ectodermal+ridge+in+limb+development.">Functions of FGF signalling from the apical ectodermal ridge in limb development.</a> Nature. 418, 501-508 (2002).</li><li>Trinh, L. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+regulates+epithelial+organization+during+myocardial+migration+in+zebrafish.">Fibronectin regulates epithelial organization during myocardial migration in zebrafish.</a> Dev Cell. 6, 371-382 (2004).</li><li>Yin, C., Kikuchi, K., Hochgreb, T., Poss, K. D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand2+regulates+extracellular+matrix+remodeling+essential+for+gut-looping+morphogenesis+in+zebrafish.">Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish.</a> Dev Cell. 18, 973-984 (2010).</li><li>Shim, K., Minowada, G., Coling, D. E., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sprouty2,+a+mouse+deafness+gene,+regulates+cell+fate+decisions+in+the+auditory+sensory+epithelium+by+antagonizing+FGF+signaling.">Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling.</a> Dev Cell. 8, 553-564 (2005).</li></ol>

The procedure of vibratome sectioning, followed by immunohistochemistry and confocal imaging allows for the visualization of the organ of Corti with minimal tissue damage. Clearly, confocal images taken from internal regions in the vibratome section show excellent preservation of cellular morphology that is limited only by fixation artifacts. Confocal images taken close to the two cut-surfaces of a vibratome section are often indistinguishable from images from internal regions, although occasional cellular disruptions, s...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Leica VT1000S Vibrating Blade Microtome</td>
 <td>Leica </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Zeiss LSM510 laser scanning confocal microscope</td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Student Dumont #5 Forceps</td>
 <td>Fine Science Tools</td>
 <td>91150-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>UltraPure Low Melting Point Agarose</td>
 <td>Invitrogen</td>
 <td>16520-050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Peel-A-Way embedding mold</td>
 <td>Polysciences</td>
 <td>18986 or 18646A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>bovine serum albumin</td>
 <td>Jackson ImmunoResearch</td>
 <td>001-000-162</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Goat Serum</td>
 <td>Invitrogen</td>
 <td>16210-064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>S100</td>
 <td>Dako</td>
 <td>Z0311</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Alexa Fluor 568 goat anti-rabbit IgG</td>
 <td>Invitrogen</td>
 <td>A-11036</td>
 <td>1:1000 dilution</td>
 </tr>
 <tr>
 	<td>Vectashield</td>
 <td>Vector Labs</td>
 <td>H-1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>YO-PRO-1</td>
 <td>Invitrogen</td>
 <td>Y3603</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>vacuum grease</td>
 <td>Fisher</td>
 <td>S41718</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Affi-Gel Blue Gel</td>
 <td>Bio-Rad</td>
 <td>153-7301</td>
 <td>&nbsp;</td>
 </tr>
 
 </tbody>
</table>

vibratome sectioning for enhanced preservation of the cytoarchitecture of the mammalian organ of corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

Watch this Scientific Journal Video about Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti at JoVE.com

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

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