Preparation of Mouse Vomeronasal Organ for Vibratome Sectioning
Transcription
Transfer the VNO to a small Petri dish under a stereomicroscope on an ice gel pack, where the remaining steps of the dissection should be performed. Next, rinse the VNO in a small amount of ice-cold S2 to prevent the tissue from drying out. Then, separate the cartilaginous capsules that contain the VNO soft tissues by grabbing the back of the vomer bone with medium forceps. For that, position the capsule for a dorsal view so that a split between both VNOs becomes visible.
Use the tip of fine forceps to separate both cartilage VNO capsules from the central bone, while keeping the vomer bone pinned down at the rear part. Make sure the medial cartilage wall that was previously attached to the vomer bone is removed.
When removing cartilage around the VNO, use forceps only at the rim of the cartilage capsule, and be very careful not to damage the delicate sensory epithelium.
To remove remaining cartilage, turn the VNO with its curved lateral side to the bottom of the dish, and securely pin down the cartilage on one side using forceps. Then, carefully move the second fine forceps from the back side at a very flat angle between cartilage and VNO to loosen the connection between tissue and cartilage.
To avoid damaging the sensory epithelium, slowly peel the VNO away from the cartilage by holding it at its caudal tip. Once the VNO is levered from the capsule, remove all the remaining small cartilage parts, as any remaining pieces of cartilage will detach the tissue from the surrounding agarose during the slicing process.
Next, place a small drop of ice-cold S2 on the first dissected VNO to prevent tissue damage. To embed the VNOs, fill both small Petri dishes to the rim with melted S3. After that, immerse each VNO in agarose, and move it horizontally back and forth several times, to remove the film of extracellular solution as well as any air bubbles from its surface.
Position the VNO vertically, with the rostral tip facing the bottom of the dish. Subsequently, place both dishes on a gel ice pack, and wait until agarose has solidified. Do not change VNO orientation once the agarose has started solidifying, as this will detach the tissue from the surrounding agarose.
In this procedure, use a small spatula to remove the agarose block from the small dish into the lid of a large Petri dish, and flip the agarose upside down, leaving the rostral tip of the VNO facing upwards. Next, cut the block into a pyramidal shape using a surgical scalpel, and take care not to damage the embedded tissue.
Then, use superglue to fix the pyramid-shaped block to the center of the bottom specimen plate, and wait about one minute for the glue to dry completely. Slice the specimen into 150 to 200 micrometer-thick sections. Briefly inspect the slice morphology under the dissecting microscope. Then, transfer all the slices to the oxygenating chamber until use.
