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

Summary

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.

Abstract

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.

Protocol

1. Isolation and fixation of inner ears

1. For analysis of cellular patterning in the organ of Corti, euthanize appropriately staged embryos or adults.
2. 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 each ear (Fig. 1A). Inner ears can be shelled out intact using forceps (such as Dumont #5, Fig. 1B). If imaging the organ of Corti, careful removal of al....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leica VT1000S Vibrating Blade Microtome	Leica Microsystems
Zeiss LSM510 laser scanning confocal microscope	Carl Zeiss, Inc.
Student Dumont #5 Forceps	Fine Science Tools	91150-20
UltraPure Low Melting Point Agarose	Invitrogen	16520-050
Peel-A-Way embedding mold	Polysciences, Inc.	18986 or 18646A
bovine serum albumin	Jackson ImmunoResearch	001-000-162
Goat Serum	Invitrogen	16210-064
S100	Dako	Z0311
Alexa Fluor 568 goat anti-rabbit IgG	Invitrogen	A-11036	1:1000 dilution
Vectashield	Vector Laboratories	H-1000
YO-PRO-1	Invitrogen	Y3603
vacuum grease	Fisher Scientific	S41718
Affi-Gel Blue Gel	Bio-Rad	153-7301