The overall aim of this procedure is to visualize the detailed morphology of drosophila, larval, dendritic, arborization neurons. This is accomplished by first collecting embryos, aging them, and generating GFP labeled genetic clones by heat shock induced lipase expression. The second step is to select larvae with fluorescent neuron clones and take an image of their dendritic ABAs.
After imaging the lava, they're dissected, fixed, and immuno stained so they can be mounted and their dendritic harbors and axonal terminal morphologies can be visualized through confocal immunofluorescence microscopy. Today I will demonstrate a protocol for morphological analysis of sula lower peripheral sensor OIDs and exons using genetic mos. This method can help answer key questions in the neurodevelopmental field, such as what are the genetic programs that control neural identity, dendritic oral shape, and external wiring?
Begin this protocol by collecting embryos on apple juice plates. The heat shock is delivered in a 38 degrees Celsius water bath, and the duration varies according to the embryo genetics and desired clone sizes. For this protocol, generate markum clones from embryos with a pand dendritic arborization neuron driver Before the heat shock, keep the marker embryos at 25 degrees Celsius, surrounded by moist and tissues for two hours.
Prepare the collection plates for heat shock by making them submersible using empty plates and para fill. Then heat shock the marker embryos for an hour to generate small clones or single neuron labeling. To increase the number of clones, use an extended heat shock by incubating for 45 minutes in the bath, followed by 30 minutes at room temperature, and then another 30 minutes in the bath.
To generate flipout clones, collect embryos for 24 continuous hours. This protocol utilizes a class four specific driver. Heat shock the flipout embryos for an hour after the heat shock.
Place the collection plates in a 10 centimeter Petri dish surrounded by moist and tissues and culture. The embryos are 25 degrees Celsius during embryo culturing monitor, and replenish the yeast paste as needed. Because nutrition critically affects neuronal development.
Maintain the culture until wandering third in star lava can be collected briefly and gently rinse the third instar larvae in tap water and transfer them to an agar plate. Now examine the larvae using a powerful fluorescence dissecting microscope and insect forceps. Identify larvae with GFP positive neurons in the body wall and transfer them to a depression slide with a drop of 80%glycerol.
Place a cover slip on the slide to immobilize the lava. Ensure that no air remains between the lava and the cover slip. Use standard confocal microscopy to visualize the neurons of interest through the lava cuticle.
To change the lava's position for an improved viewing angle, gently push on the cover slip to roll the lava around. Begin this protocol by transferring a lava to the SIL guard plate using insect forceps. Then pin it down at the anterior and posterior ends.
Then add calcium free HL 3.1 buffer. Now cut halfway through both ends of the lava perpendicular to the anterior posterior axis. Then cut along the midline between the two trachea.
Carefully cut each individual tracheal connection to the body wall before gently removing the gut. This keeps the segmental nerves running between the PNS and the CNS intact. With the larva still pinned to the cell guard plate, fix it in 4%Para formaldehyde in PBS for 20 minutes of room temperature on a gently rotating shaker, perform each dissection in less than five minutes.
Otherwise, the nites of the dendritic authorization neurons will begin to degrade. After the fixation, remove excess larval tissues. Then wash the fillets three times with PBST for 10 minutes per wash.
After the washes, incubate the larvae for 20 minutes of room temperature in blocking solution with gentle shaking. After the block is complete, replace the blocking solution with the primary antibody solution. Place the sample in the small plastic container surrounded by moist and tissues and incubate it overnight at four degrees Celsius The next day, continue the incubation for an additional hour at room temperature.
Then wash the lava fillet six times in PBST for 10 minutes per wash. Now add the secondary antibody and put the fillets in a dark container to prevent photobleaching of the Fluor. Incubate the fillets for either hours at room temperature or overnight at four degrees Celsius, followed by one hour at room temperature.
Finally wash the fillets six times in PBST for 10 minutes per wash. The tissues are then ready to be mounted. To visualize axon termini, simply mount the larval fillet cuticle side down in 80%glycerol in a depression slide.
To make clearer images of dendrites, make a permanent mount with PLL coated cover slips. To code the cover slips tho and aliquot of PLL. Bring it up to 10 milliliters of double distilled water and add 20 microliters of Kodak photo flow.
Submerge the cover slips for 30 minutes in the PLL solution and air dry them upon removal. Then briefly rinse them with water and air. Dry them again.
Repeat this process twice. Beginning with the PLL bath, the treated cover slips will last approximately one month. Now mount a dissected larval fillet muscle side down into a drop of PBS on a PLL cover slip.
The fillet will adhere quickly to the cover slip using a piece of tissue paper. Remove as much liquid as possible, however, do not allow it to completely dry out. Next dehydrate larval fillets, and a series of 10 minutes ethanol baths, beginning with 35%ethanol, followed by 50%then 70%then 95%then 100%and finally a second, 100%ethanol bath.
Follow the ethanol baths with two or three 10 minute baths in xylene. Then put a drop of DPX on a clean slide and carefully set the cover slip into the DPX, fill it side down. Keep the slide in the dark and wait one day for the DPX to set.
Before imaging the tissue using in vivo and focal microscopy, it is possible to capture images like this. The entire arbor of a Class four dendritic arborization neuron. This is a closeup of a dendritic arbor from an IHC labeled class three neuron that has been correctly fixed to preserve morphology.
The inset shows the degradation that can occur after an unsuccessful dissection and fixation. This overlay shows a single class four axon terminus in the CNS. The axon is labeled with anti GFP in green and all class four termite a co stained with anti CD two in magenta.
While attempting this procedure, it is important to remember to dissect and fix samples quickly by tracing axons from the periphery to the CNS. It is useful to keep in mind that PNS neurons in each body will segment project to the cognitive segment of the vent nerve code. Also, if you only want to visualize dendrites, remove the CNAs during dissection, and staining can be carried out in append of tubes instead of on plates.
When mounting these filets for dendrite imaging first cut off the head with mouth hooks and posterior with sparkles. For a pleasure preparation.
