全山解剖和成年小鼠耳蜗的免疫

The overall goal of this whole mount dissection method is to isolate the sensory region of the calcified adult cochlea as three intact turns:the apex, middle, and base. This dissection method preserves the three dimensional structure of the organ of Corti, and allows visualization of all cells when combined with immunostaining and confocal microscopy to image at different planes in the Z axis. Compared to previous dissection methods, we use a different strategy that generates three cochlear turns that are larger and less likely to be lost or damaged in the immunostaining process.

Visual demonstration of this method is critical as the dissection is difficult to learn. Because the organ of Corti is fragile, there is a small margin for error when removing the spiral ligament. After euthanizing the animal according to approved procedures and removing the brain, begin by identifying the temporal bones in the base of the mouse skull.

Then scrape away the cranial nerves using standard pattern forceps. Place the forceps at the tip of the otic capsule and use the thumb of the opposite hand to press down on the posterior semi-circular canal to dislodge the encapsulated cochlea. Free the bottom half of the temporal bone from the skull either manually with the thumb and index finger or using 10.5 centimeter fine scissors, and place in methanol free electron microscopy grade 4%paraformaldehyde to fix.

After fixation, decalcify the temporal bones according to the instructions in the written portion of the protocol. To determine if the sample is adequately decalcified, place the temporal bones on a silicone-elastomer coated dissection dish, and gently press forceps onto the snail-shaped cochlea. If the tissue is spongy, then decalcification is complete.

Add PBS to the dissection dish, and then while working under a stereo dissection microscope, use number four or number five forceps to hold the temporal bone in the vestibular region and five milliliter Vannas-Tubingen spring scissors to cut away excess otic capsule tissue along the sides and above the apex. Using 2.5 milliliter Vannas spring scissors, insert one blade into the oval window and make several small cuts along the spiral ligament of the basal turn. Now, insert one blade of the five milliliter Vannas-Tubingen spring scissors into the region just cut, and place the other blade on the outside of the temporal bone, medial to the oval window.

This cut separates the basal turn from the middle and apical turns. Next, to complete the dissection of the basal turn, use the 2.5 milliliter spring scissors to cut the spiral ganglion nerve fibers that connect to the modiolus to release tension in the basal turn. Cut below the basal turn to separate from the vestibular organs.

Use forceps to guide the tissue and to pin the spiral ganglion fibers to the silicone-elastomer coated dish. Make a series of small cuts to remove the spiral ligament from both above and below the organ of Corti. With the spiral ganglion fibers pinned to the silicone-elastomer coated dish, use the forceps to remove any remaining Reissner's membrane from the organ of Corti.

Make several cuts to reduce the thickness of the spiral ganglion axons. Then grasp the remaining axons of the spiral ganglion with forceps and transfer the dissected basal turn to a 48 well plate containing approximately 500 microliters of PBS. Now separate the middle and apical turns by first placing the remaining two thirds of the cochlea apical side down.

Then insert one blade of the 2.5 milliliter scissors into the scala media, where the middle turn was previously connected to the basal turn, and make several cuts along the spiral ligament of the middle turn. Using the five milliliter scissors, insert one blade into the cut region, with the middle turn placed on top of the blade. Place the other blade on the outside of the bony labyrinth, at a 90 degree angle from the apical tip.

This cut separates the middle turn from the apical turn. Next, complete the dissection of the middle turn by removing the spiral ligament from the organ of Corti. Transfer the dissected turn to a 48 well plate, as before.

Now, use 2.5 milliliter Vannas spring scissors to open the cap that covers the apical turn, and repeat the procedure to remove the apical turn spiral ligament from the organ of Corti. Transfer the dissected turn to a 48 well plate, as before. To begin the immunostaining procedure, first aspirate PBS from each well, then replace with 200 to 300 microliters of blocking solution and incubate for one hour at room temperature on a 3D rotator.

Once the incubation time has elapsed, remove the blocking solution and replace with 100 microliters of primary antibody, diluted appropriately in carrier solution. Incubate overnight at four degrees Celsius on a 3D rotator. The next day, remove the primary antibody solution and wash each well with 500 microliters of PBS for at least five minutes each time, at room temperature.

Repeat the wash twice. After the last wash, aspirate the PBS and be careful not to suck up the dissected turn in the pipette tip. Replace with 100 microliters per well of fluorescently tagged secondary antibody diluted in carrier solution.

Place the 48 well plate in a black box to protect from light, and place on the 3D rotator to incubate for two to three hours at room temperature. Following the incubation, aspirate the secondary antibody solution and wash three times with PBS as before. After removing the last wash, add 100 microliter of Hoechst to label nuclei.

Protect from light and incubate for 15 to 20 minutes at room temperature on a 3D rotator. Finally, remove the Hoechst solution and wash three times with PBS as before. After labeling each slide, pipette 50 microliters of mounting media onto each slide.

Then using number four or number five straight jeweler's forceps, grasp the axons of the spiral ganglion and gently transfer one cochlear turn from the 48 well plate to the mounting media. Use the microscope to ensure the dissected turn is not folded or twisted. Place one end of a cover slip on a slide, and gently release to let the cover slip fall.

Use a stereo dissection microscope to ensure that the cochlear turn is not located near an air bubble. If this occurs, gently move the cover slip back and forth to reposition the cochlear turn. Repeat the procedure for the other cochlear turns.

Mount one cochlear turn per slide to prevent light exposure and photo bleaching during the imaging process. Place slides in a slide folder so that they lie flat. Protect from light and allow the mounting media to cure overnight at room temperature.

The next day, seal cover slips with clear nail polish, and store at room temperature or 20 degrees Celsius until imaged. This confocal slice image shows the cochlear middle turn isolated from a p15 mouse. 420X images were overlaid to reconstruct the whole middle turn.

The hair cells, labeled with a rabbit anti-myosin VIIa primary antibody and an Alexa 647 conjugated secondary antibody appear magenta. Supporting cells, labeled with a goat anti-SOX2 primary antibody and an Alexa 568 conjugated secondary antibody appear green. Nuclei appear blue due to Hoechst stain.

The scale bar represents 100 microns. This image shows an optical cross section of the middle turn isolated from a six week old mouse in the X, Z plane. Again, the hair cells are labeled with a magenta fluorophore and the supporting cells with a fluorophore that appears green.

This is an increased magnification of the previous image, with the crosshairs removed. The scale bar represents 20 microns. The following four images show some of the problems that can occur during whole mount dissection, or when mounting cochlear turns on slides.

Here, on the left side of the image, the cochlear tissue was cut next to the last row of outer hair cells, causing many of these cells to be mounted at varied angles. In this image, a section of the organ of Corti on the left side has been cut off. There is a hole punched in the outer hair cell region in the middle of the image.

Here the organ of Corti is folded in several places. Once mastered, the whole mount dissection technique can be completed in 20 to 30 minutes. While performing this procedure, it is important to avoid placing the forceps on the organ of Corti and to avoid pulling or grasping on the spiral ligament.

Instead, close the forceps and pin the spiral ganglion fibers to the silicone-elastomer coated dish. Samples from one to three week old mice are more forgiving and useful to learn the dissection method. In addition, samples with hair cell damage are more difficult to dissect, with noise exposed tissue especially difficult and fragile.

Following this dissection procedure, other methods, such as scanning electron microscopy, can be performed. However a different fixative is needed. In addition, we have made slight modifications to this protocol for dissection of rat cochlear tissue, and in the future, we hope to modify further for dissection of cochlea from chinchilla, gerbil, and guinea pig.