Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The overall goal of this procedure is to provide a method for on face visualization of ciliary proteins in the olfactory system of mice. This is accomplished by first splitting a urine skull parallel to the suture line. The second step is to carefully expose the olfactory epithelium covering the nasal septum.

Next, the olfactory epithelium is isolated from all connections to the head and transferred to an adhesive glass slide. The final step is a short fixation and gentle handling during staining of the tissue. Ultimately, immunofluorescence microscopy is used to show protein localization in the olfactory clia.

The main advantage of this technique over existing methods like cryo sections, is that of fast preparation. Provide additional details when analyzing proteins in the Celia of olfactory sensory neurons. Visual demonstration of this method is critical as the dissection steps are difficult to learn because the ciliary structures can easily be damaged leading to inconsistent results.

Begin with the freshly decapitated mouse head. Start the dissection by removing the skin to expose the skull and nose with the paper towel. Wipe away remaining blood and tissue.

Then dissect away the lower jaw and the front teeth next to separate the septum from the maxilla in size, the dorsal bone bilaterally, one to two millimeters parallel to the suture line. Then with a single cut, split the nose without touching the septum. Carefully remove any remnant bones and terminates, thus exposing the septum.

Now, remove the nasal bone using the curved tip of the forceps, insert the tip between the epithelium and nasal bone. Apply a slight pressure on the bone and lift it up to remove the bone to gain access to the septum from either cavity. Carefully remove the remaining maxilla.

If the animal is older than P 28, then the tip of the frontal bone covering the olfactory bulbs must now also be removed. The olfactory epithelium has a yellow color while the bordering respiratory epithelium is white and on it, the movement of Celia can be seen cut along the border between these epithelial with fine spring scissors. Then extend the cut to separate the septum from the ventral connection to the vulner bone.

Then cut along the border between the septum and lamina cosa of the seth. Start with placing an adhesive glass slide onto the edge of a Petri dish filled with ringer's solution. Submerge half the slide with the tines of the forceps.

Carefully transfer the ethmoid bone with the olfactory epithelium to the dish. Do not use the forceps tips. Now grab the perpendicular plate of the ethmoid bone with one forceps.

With the second forceps. Carefully peel the epithelium off one side of the septum by sliding the tip between the plate and the epithelium and lifting away. The epithelium should stay positioned with the ciliary side up in case the epithelium flips and rolls up.

Identify the ciliary side by comparing the unroll tissue shape. With this one without touching the ciliary side. Grab the epithelium at the border and tug it up onto the glass slide.

Now rotate the septum and peel the epithelium off the other side. Once both pieces of epithelium are on the slide, remove the slide from the dish. Then using a paper towel, wick away the solution surrounding the tissue.

Once dried, draw a circle around the tissue with a liquid blocker pen. Now apply 150 microliters of fixative indirectly to the tissue and wait 10 minutes. This short time will fix Cecilia and work for a variety of antibodies.

However, the whole epithelium will not be fixed, so it must be imaged shortly After staining. After 10 minutes of fixation, carefully aspirate the fixed solution without disturbing the tissue with unnecessary mechanical forces. Wash the tissue twice with PBS.

Never apply solutions directly onto the delicate clia. Then apply the blocking buffer and let the tissue incubate for an hour at room temperature. Meanwhile, dissolve the primary antibody in blocking buffer at the appropriate concentration and centrifuge for five minutes to remove any precipitates After the blocking, apply the primary antibody dilution to the epithelium and put the slide in a humidified box.

Transfer the box to four degrees and let it incubate overnight. The next day. Use three five minute washes with PBS to remove the excess primary antibody.

Then apply the secondary antibody prepared like the primary antibody. Let the tissue incubate for an hour at room temperature. Now, it should always stay in the dark after an hour.

Use three washes with PBS to remove the excess antibodies. Now, remove the circle of liquid blocker with a razor blade or a paper towel. Wash the epithelium one last time for five seconds using distilled water.

Then mount the tissue in anti fade mounting reagent and visualize it as soon as possible. Using the described on face preparation of olfactory epithelium, the expression of keel two, A molecular shown to play a role in Axon guidance in the olfactory system was investigated unambiguously keel two localizes to the Celia. In a subset of sensory neurons in cryo sections of the same tissue, the localization of Kyl two was difficult to discern.

Another protein was then investigated the olfactory receptor, M-O-R-E-G, which is known to have strong ciliary localization as clearly seen in cryo section preparations. Using the MASE approach, it is possible to identify individual Celia with neurons expressing the receptor. This marks an exceptional improvement in detail over cryosectioning.

It was also possible to look at the localization of proteins at different age stages, such as at the prenatal stage. Using this on face tissue preparation, Right. While attempting this procedure, it is important to remember that any dispensable touching and squeezing off the epithelial must be avoided to preserve the fragile ciliary structures.

After watching this video, you Should have a good understanding of how to perform reproducible or fast preparation for a whole mount olfactory CLIA tending to get an additional perspective onto the olfactory epithelium.