The overall goal of the following experiment is to visualize the olfactory receptor neurons using whole mount antennae staining. First anesthetize the fly to cut its head vertically. Next, place the dissected fly head on an apple plate.
Then cut the third segment of the antenna and place it to the glass bottom culture dish in order to perform immunohisto chemistry. After mounting the antennae in a slide glass, we can visualize the diverse types of olfactory receptor neurons through confocal Microscopic hi. We are working On do olfactory system.
The olfactory system is used to distinguish between an immer variety of other molecules. The wide range of distinct olfactory neurons that exist within this system allows for the precise and coordinated transmission of information from target receptors to neurons within the brain. Today we'll describe the whole mount antenna staining to detect the diversification of these olfactory receptor neurons.
In order to ensure a successful whole mount and antenna staining, it is critical to prepare a fresh batch of fixative immediately prior to beginning the protocol. Anesthetize the fly And then cut the fly head vertically by holding its body with forceps. Now, carefully place the dissected antenna on an apple plate.
Cut the third segment of the antenna using fine dissection scissors. Place 90 Microliters fixative solution in the center of the dish. Using a sharp needle, gently transfer the dissected antenna directly to the Fixative solution.
Now allow the fixation to proceed undisturbed for 40 minutes at room temperature to end the fixation. Wash the antennae three times with PBST for 10 minutes per wash during removal of the washing solution. First, bring all the antennae to the center of the dish using a needle.
Then remove the solution from the side of the dish. Agitation during incubation is Unnecessary. To prepare for Staining, remove the last wash of PBST and block the fixed antennae by filling the chamber with 90 microliters of 5%horse serum in 0.1%PBST replace the blocking solution with primary antibodies diluted in a mixture of 5%horse serum.
In PBST, the primary antibodies used are mouse anti GFP one in 400 and rat anti eof, diluted to a concentration of one in 200 to prevent the antennae samples from drying out. Place a moist tissue into the lid of the culture dish and wrap it with the paraform. Then place the culture dish in a plastic container containing moist tissues.
Incubate the antennae in the primary antibodies for 48 hours at four degrees Celsius after 48 hours. Clear the unbound primary antibodies with six washes in PBST for 10 minutes per wash. Similar to the previously described fixative removal procedure.
After an hour of PBST washes, add secondary antibody solution diluted in a mixture of 5%horse serum In PBST in this example, the antibodies are SI two anti rabbit IgG and SI three anti rat IgG. Cover the container with aluminum foil from this point forward to prevent fluoro four. Photobleaching next incubate the antenna in the secondary antibody for 48 hours at four degrees Celsius.
At this point, the antenna is now ready for mounting to mount the antenna. Remove as much of the PBST from the culture dish as possible, and gradually begin to introduce glycerol to the antennae. Subsequently, add 40%glycerol to the dish for one to two minutes, then remove it and add 80%glycerol and leave it for five minutes.
Fluorescent staining revealed Elof positive neurons and the targeted olfactory neurons. By combining this procedure With other similar methods, including the generation olfactory neuron genetic mosaics, we hope to initiate the mechanical controlling olfactory neuron de diversification and answer other important scientific questions.
