In the inner ear of zebrafish, there are three pairs of sac, like end organs, the utricle, the S, and the Lana. The utricle locates most anteriorly while the S and the lega closely attached to each other and locate posteriorly to the utricle After gross dissection, the S and lega stay attached and the stonelike structure otolith can be easily seen in each end organ in the following fine dissection. The S and lega are separated from each other by cutting from the ventral side.
Otoliths are removed and the nons sensory epithelial tissues are trimmed off to get sensory epithelial tissues from all three end organs. Hi, I'm Jean Leon from the Lab of Sean Burgess in the developmental genomics section of the National Human Genome Research Institute. Today we'll show you a procedure for the fine and grass dissection of the inner year sensory epithelia from the adult zebra zebrafish.
We use this process in our lab to study the gene expression in the inner year sensory epithelia. So let's get started. To begin the dissection, euthanize the adult zebra fish with buffered MS 2 22 0.03%decapitate the fish, then cut off the lower jaw.
Next, place the head on the dissection pad with the ventral side up and immobilize the tissue with insect pins under the dissection scope. Remove the soft tissue ventral to the skull with fine tweezers using the same tweezers. Crack open the ventral part of the skull capsule.
Finally, fix the fish head in a small plastic container in 4%Para aldehyde in one XPBS at four degrees Celsius overnight the following day. Discard the para formaldehyde because para formaldehyde is considered hazardous waste. Please remember to dispose the used para formaldehyde solution according to the lab safety regulations of your institution and rinse the fixed fish head in one XPB S3 times five minutes each time.
Now that the fish head is fixed, gross dissection of the inner ear can begin. Start by placing the fish head on the dissection pad with the ventral side up. Immobilize it with insect pins.
From this point forward, the rest of the work will be done under the dissection scope. With a pair of fine tweezers, remove the skull bone over the inner ear and then carefully pull out the inner ear tissues, the utricle, the S and the lega. The s Saul and lega are tightly attached to each other and thus pull out together while the utricle anterior to the sial and the gaa usually detaches from the other end organs during dissection.
Last, put the inner ear tissues in one XPBS following the gross dissection. One can proceed with fine dissection of the inner ear sensory epithelia. The fine dissection is carried out on a clean glass slide in a drop of one XPBS.
Use two pairs of fine tweezers to remove the U otolith from the utricle with a pair of fine tweezers and a needle blade trim off the neurosensory epithelial tissue and the innervation from the epithelial place. The S and lega, which are still attached to each other and the PBS drop with the ventral side. Up next, remove the leg gainor oli.
Use the needle blade to carefully cut in between the sial and the gaa. Finish by trimming off the excessive neurosensory epithelial tissue in the innervation with fine tweezers and a needle blade. The sensory epithelia are now ready for staining.
To begin the staining, rinse the sensory epithelial with PBT for three times at five minutes each time. Next, incubate the epithelial with Alexa Fluor 4 88 fain at a one to 1000 dilution for 30 minutes. At room temperature, the Phin binds to actin filaments in the cells as the a pickle bundle of the hair cell and the sensory epithelia as an actin rich structure.
It will be strongly stained by the Phin. Rinse the sensory epithelia with PBT for three times at 10 minutes each time. To view the cells, mount the epithelia onto a slide with vector shield Hard set mounting medium with DAPI check the stained tissue under the fluorescent microscope with FITC filters After successful dissection and staining, intact epithelia with strong hair cell bundle staining can be seen under the microscope under lower magnification on the fluorescent microscope.
The most distinguishable structures are the hair cell bundles, each of which appears as a bright. in the sensory epithelia. It is the most strongly stained structure because it is very rich in acton.
The bundle is a unique atypical structure of the hair cell that can be used to easily identify hair cells as well as identify the regions of the sensory epithelia. We've just shown you how to dissect the inner year sensory epithelial from the adult zebrafish. If you skip the step of fixation, the procedure can also be used for preparing tissues for the extraction of D-N-A-R-N-A and proteins.
So that's it. Thank you for watching and good luck with your experiment.
