The overall goal of the following experiment is to image the organ of corti with maximal preservation of cellular architecture. This is achieved by sectioning, dissected and fixed inner ears with a vibrato to obtain thick sections while minimizing the number of cut surfaces per cochlear. The Vibram sections are then stained by immunohistochemistry, which allows the identification of various cell types and cellular structures in the organ of corti.
Next, the sections are mounted and imaged by confocal microscopy in order to obtain high resolution images that define Z positions Throughout each thick Vibram section results are obtained that allow for the confocal microscopy of the cells and structures in the organ of corti with minimal disruption by the sectioning process. The main advantage of this technique over existing methods such as paraffin and cryosectioning, is that physical damage to the cochlea is minimized, allowing better preservation of the cellular architecture of the organ of corde In order to isolate the inner ear. First, decapitate a euthanized adult mouse using a razor blade.
Open the skull along the midline and then remove the brain to expose each ear using forceps carefully, shell out both inner ears. Immerse the inner ears in a cryo tube vial of fresh 4%para formaldehyde, and gently rock it overnight at four degrees Celsius. The next day, wash out the fixative with three changes of one times phosphate buffered saline, decalcify adult tissue by incubating in 10%EDTA at four degrees Celsius for five days.
Rinse and store the inner ears in one times PBS until ready for processing. Make 4%low melting aros by adding the aros to one times PBS and microwaving until the aros is in solution. Keep the molten aros in a 55 degrees Celsius water bath until ready to use.
Remove the inner ear from the PBS using forceps and position it at the bottom of a peel away. Embedding mold, pipette away any remaining liquid, and then fill the mold with mol and aros completely covering the inner ear to generate cross sections through the organ of corti. Position the inner ear so that it is viewed laterally with the lateral semicircular canal.
Visible angle, the inner ear so that the line formed by the posterior most region of the cochlear is at a 45 degree angle from the intended plane of sectioning. Once the agros has solidified, peel away the plastic mold. Use a razor blade to trim away excess aros and create a cube.
Making sure to leave enough surrounding aros to keep the tissue well supported. During sectioning, attach the base of the agros cube to the Vibram cutting surface using super glue section at 40 to 100 micron thickness and collect the slices in a dish of one times PBS with 0.1%Triton X 100. Using forceps.
Dissect the aros away from the tissue sections. Transfer the sections to a dish your fresh one times PBS with 0.1%Triton X 100, using a pipette tip with a wide opening and proceed with the antibody staining in a tissue culture plate. After the antibody staining is complete, tissue sections are mounted onto a microscope slide.
To prevent crushing the thick tissue sections, place a layer of vacuum grease in a square around the section to create space between the cover slip and the slide. Transfer the tissue section onto a microscope slide. Press down the edges of the cover slip firmly to create a seal.
Alternatively, pipe ETA one microliter dot of aros chromatography, resin of known mesh size in each of the four corners where the cover slip will rest, lay down the cover, slip and seal the edges with nail polish before imaging in the first image of a confocal Zack through an organ of cor T section, the inner hair cells adjacent inner hair cells, outer hair cells, and dieter cells are visible. When nuclear staining, the pillar cells are visible by S 100 antibody staining and appear yellow. As the Zack progresses through the section at three micron steps, the number of pillar cells can be counted by noting the appearance and disappearance of their nuclei.
After watching this video, you should have a good understanding of how to section the inner ear by vibram, perform immunohistochemistry on these sections, and then image by confocal microscopy. This simple procedure which is generalizable to any antibody ensures maximal preservation of the cellular organization of the organ of corde.
