To study whether defects in long distance axonal transport are present in neurological diseases such as Alzheimer's disease or Huntington's disease. Axonal transport of transport vesicles is imaged in live esophagal lava. Here wondering.
Third in star larvae that express A GFP tagged physical protein are dissected on a gel cube, then transferred to the microscope for in vivo imaging. Fluorescence microscopy is then used to track sical transport within the axon. The main advantage of using this technique over existing methods is to visualize live undisrupted movement of synaptic vesicles and larval axons.
Prepare for the dissection by ensuring that the following supplies and reagents are at hand. Dissection buffer. A Sill guard cube cut to one inch wide, one inch long, and one centimeter high placed on a glass slide.
Extra short dissection pins. The sharp bottom portion of manum pins works best in dissection. The correct length can be attained by inserting longer pins into the gel and cutting off any portion of the pin that protrudes.
You will also need one pair of sharp microdissection spring scissors, and two sets of fine tipped forceps. Use A small metal spatula to transfer wandering. Third in star larvae expressing A GFP tagged SLE protein to a Petri dish.
Next, apply deionized water to the larvae in the Petri dish to get rid of all the food. Then using forceps, transfer Them to a dissecting gel cube to perform The live dissection. Use forceps to transfer a larvae to the dissection gel cube.
On a glass slide, add dissection buffer to the slide to immerse it. Next, pin the lava at the anterior and posterior ends with the dorsal side up. Using microdissection scissors, make a small cut at the midline near the posterior end.
From this incision, make another cut through to the anterior. The gut intestines and fat bodies will ooze out using two pins. Fix the cut cuticle the lateral edges to the sil guard cube.
Use a pipette to add a drop of dissecting buffer. Then using forceps carefully remove the gut intestines and fat bodies. Apply a kim wipe to one side of the lava to remove the buffer while simultaneously adding fresh dissecting buffer with a pipette.
The lava should never be dry. To label mitochondrial or lysosomal structures in the lava, replace the dissecting buffer with dissecting buffer containing mito tracker or lyo tracker. Place the dissecting cube in a square humidified chamber and cover it in aluminum foil.
Incubate for 10 minutes at room temperature. After the incubation of the in vivo marker, repeatedly soak up dissection buffer surrounding the larvae and add more dissection buffer with a pipette. Repeat this five times to wash off any remaining marker.
After replacing the last wash, push the pins completely into the S guard and immediately place a cover slip to avoid larva movement. The larva Is now ready for observation To perform live imaging of the dissected lava. Place the sample onto the stage of an inverted microscope to image tagged vesicles.
Within larval segmental nerves. Perform imaging using a 100 times objective lens. If robust, GFP staining is observed in the cell bodies in the ventral ganglion image, the segmental nerves using a dual view system with filters for C-F-P-Y-F-P or R-F-P-Y-F-P and split view software two in vivo tagged proteins can be viewed simultaneously.
In this time lapse series are many axonal vesicles on many axonal tracks within a larval segmental nerve. Note that GFP representing axonal accumulations also note the bidirectional movement of vesicles. Here, the movement of axonal vesicles on a single track within a larval segmental nerve are seen.
Note, the bidirectional movement movement of these sles are on average one micron per Second. After watching this video, you should have a good understanding of how to complete a clean dissection for in vivo viewing of GFP tagged vesicles and gsof larval axons.
