Axonal transport can be studied by visualizing the larval segmental nerves. A third instar drosophila. The larva is first dissected, exposing the CNS without damaging the optical lobes, ventral ganglion, and segmental nerves.
The second step of the procedure is to fix the lava in formaldehyde before incubating with primary antibody overnight and the secondary antibody for an hour in the dark. The lava is then mounted on a glass slide and axonal transport defects. Protein localization and protein distribution can be examined through immunofluorescence microscopy.
Though this method can provide insight into axonal transport, it can also be applied to other systems such as synapse and muscle morphology. Generally, individuals new to this method will struggle because initially it's difficult to perform this allowable dissection, especially to keep the brain segmental nerves and muscles intact. To begin this procedure, prepare one times dissection buffer fixative, one times PBT, and 5%BSA solution.
As detailed in the table shown before the dissection, gather a clean pair of number five biceps, a clean pair of straight blade fan scissors and pins to start the dissection, use a micro spoon to collect wandering. Third in star larvae with a specific genotype onto a Petri dish. Then wash the larvae by rinsing them in a Petri dish filled with deionized water to get rid of all the food.
Next place a lava on the S guard dish with the dorsal side up. Pin the lava at its anterior and posterior. Ends place a drop of the one-time dissection buffer on the pinned lava using fine scissors.
Nip the lava on the dorsal midline near the posterior end. Using the scissors, cut the lava cuticle from the posterior end through to the anterior end. Place a drop of new one times dissection buffer onto the dissected lava.
Using a pin, pick one lateral edge of the cuticle near the middle of the lava and pin the cuticle using another pin.Pin. The second lateral edge of the cuticle, two pins are used on each lateral side as shown in the diagram. Now, carefully remove the intestine and fat bodies and traia leaving only the brain with the two optical lobes and ventral ganglia.
With the segmental nerves attached fixation and antibody staining are next performed with the larvae attached to thes guard dish. Incubate the dissected larvae in 50 microliters of fixative for 30 minutes. Replacing the fixative every 15 minutes after fixation, rinse the larvae in one times PBT for 30 minutes.
By replacing the buffer every 10 minutes, it is important to note that the larvae should always be submerged in buffer. Prepare the primary antibody solution by diluting it to the appropriate concentration in the 5%BSA solution. Optimal concentrations differ for different antibodies.
Construct a humid chamber by lining a plastic dish with Kim. Wipe soaked in water. Replace the last one times PBT.
Rinse with the prepared primary antibody solution and incubate in the humid chamber at four degrees Celsius overnight the following day. Rinse the dissected larvae in one times PBT for 30 minutes by replacing the buffer every 10 minutes. Prepare the secondary antibody by diluting it to the appropriate concentration in the 5%BSA solution.
And wrap the humid chamber in foil as some secondary antibodies are light sensitive. Incubate the larvae with the secondary antibody solution at 25 degrees Celsius for an hour. When the incubation with the secondary antibody is done, rinse the larvae in one times PBT for 30 minutes by replacing the buffer every 10 minutes and proceed to mount and visualize the dissected larvae before mounting the lava on the slide.
Prepare the slide by spreading two vertical lines of nail polish in the middle with a space large enough for a cover slip to sit on as shown in the diagram. Let the nail polish dry completely when the nail polish has dried. Place a drop of mounting buffer in the space between the vertical nail polish lines on the slide as shown in this image.
The slide is now ready for mounting. Retrieve the larvae after the last one times PBT. Rinse and gently remove the lateral pins from a lava.
Be careful not to tear the cuticle carefully. Remove the two remaining pins and pick up the lava with forceps and place it on the prepared slide. Make sure the lava is not flipped over and is oriented ventral.
Side up gently place a cover slip on top and seal the edges with nail polish as illustrated here. The larvae are now ready for visualization under a fluorescent microscope. This representative image is of a wild type lava stained with antibodies against the synaptic vesical protein, cystine string protein.
The lava segmental nerves are smoothly stained.Here. A motor protein mutant lava was stained with antibodies against CSP. The segmental nerves show massive accumulations that stain brightly for CSP.
While attempting this procedure, it's important to remember to retain the segmental nerves attached to the brain and to keep the muscles intact. After watching this video, you should have a good understanding of how to dissect. Fix an immunostain third and start larval segmental nerves to study axonal transport using a fluorescent microscope.
