The overall goal of this procedure is to visualize proprioceptive cortone organs in larvae and puby of drosophila. This is accomplished by first rearing and collecting third in star larvae or puby after dissection of the larvae or pupi. They're fixed for immuno staining.
Next, the fixed larvae or pupi are immuno stain with the appropriate antibodies. The final step is to mount the stained samples for microscopy. Ultimately, confocal microscopy is used to visualize the cortone organs.
This method can help answer key questions about post symbionic development and patterning of prop receptors in oph. Although this procedure may provide insight into prop receptor development, it can also be applied to other organs such as other types of sensory organs and tendons. First, assemble the necessary tools, solutions, and a vial of wandering.
Third in star larvae, keep the PBS and FIXATIVE solutions on ice. Pick a wandering third in star larvae from the vial wall and place it in a 50 microliter drop of ice cold PBS on a dissection plate made of cigar elastomer in a tissue culture dish. Using a dissecting microscope holds the larvae dorsal side up near the mouth hooks with fine forceps and stick an insect pin through the brain.
Grab the posterior end of the larvae with the forceps. Pull gently and stretch the larvae gently lengthwise. Stick another insect pin between the two posterior sphericals using spring scissors.
Cut two horizontal incisions in the body wall perpendicular to the anterior posterior axis, keeping close to the anterior and posterior pins. Next, cut the larval body wall along the dorsal midline from the anterior to posterior incision. Remove the internal organs and the trachea using fine forceps.
Then wash gently once or twice with PBS. Grab the two body wall flaps with forceps. Stretch them out and pin them to the plate using two insect pins for each side.
Remove the PBS and add 50 microliters of fixative solution. Incubate for 20 minutes at room temperature. After removing the fixative and washing twice with PBS, pull out the insect pins using spring scissors.
Cut the larval head and tail leaving a rectangular filet. Transfer the fixed tissue to a chilled einor tube with PBS. Once a sufficient number of larvae are fixed, one can continue directly to antibody staining.
If immediate staining is not desired, the fixed larvae should be washed three times at five minutes each with 95%ethanol and then stored in the ethanol at minus 20 degrees. Attend to the vials as for larvae fixation until third instar larvae begin to ate. Examine the vials every hour and mark larvae that have ated.
Allow the marked pube to develop for 30 to 40 hours at 24 degrees Celsius, or 24 to 27 hours at 29 degrees Celsius using fine forceps. Pick 20 to 30 pube of the appropriate age and put them in a dark porcelain multi-well dissecting dish. Be careful not to damage the tissues of interest.
Next, extract the pupi from their pupil cases. Begin by peeling off the operculom and continue until the pupa is free. Extract all the pupi and put them into a well filled with PBT.
Place five puy on a flat surface between the wells of the dissecting dish using a micro dissection knife. Cut the tip of the head and the posterior tip of the abdomen. Hold the pupa in place using fine forceps and use a one milliliter syringe to wash out the internal organs of the pupa by injecting PBT through the anterior opening.
Wash the dissected pupa briefly by dipping it in a well filled with PBS and move it into the cold fixative in an einor tube kept on ice. Incubate the puy overnight at four degrees Celsius. The next day.
Discard the fixative and wash the puby with PBT three times for five minutes per wash. Keep the wash puby on ice. Fill two wells of the dissecting dish with PBT.
Using a polyethylene pasti pipette. Transfer several puby to one of the wells. Then move one pupa at a time into the second.Well.
Using two pairs of sharp forceps with perfectly aligned tips, secure the pupa to the bottom of the well and then gently tear the cuticle. Removing the wing cuticle is the most difficult aspect of this procedure. We always lose some of the wings during this step, which is why we start the procedure with a relatively large number of pupa.
Once the cuticle is torn, peel it off the wing. Being careful not to disconnect the wing from the pupa. After peeling off the cuticle from the wing blade, continue peeling the cuticle of the wing hinge.
Once the wing cuticle has been peeled, one can attempt peeling off the leg cuticle in a similar manner. Many legs are likely to be lost in the process, but despite the low yield, this step is highly recommended since it greatly improves the staining of leg cortone organs. Place the pupa in an einor tube filled with methanol and keep it at room temperature.
Continue peeling off the cuticle of all the puy and add them to the same tube for a total of at least 10 nicely peeled puy. For each staining, remove the methanol and wash three times five minutes each with 95%ethanol. Fixed puy can be kept for long periods of time in 95%ethanol at minus 20 degrees prior to immuno staining.
To mount the larvae, place a drop of mounting media on a microscope slide and then position the larvae with the cuticles facing down and body wall muscles facing up. Put a small drop of mounting medium on a clean cover slip and use it to cover the preparation without applying any pressure on the mounted larvae. To mount the puby.
First, prepare two microscope slides with a drop of mounting medium, one for dissection and the second for mounting. Place several pu on one of the slides and then use the spring scissors to remove the head and the posterior part of the abdomen. Next, separate the dorsal half of the thorax from the ventral half by cutting between the wings and the legs.
Place the dissected body parts of the pupa in the drop of mounting medium. On the second slide, ensure that the wings and legs are stretched out and try to minimize overlaying of tissues as much as possible. Finally, place a drop of mounting medium on a cover slip and place it over the sample without applying excessive pressure.
This confocal micrograph of immuno stain cortone organs in a third in star larvae shows a nicely stretched abdominal segment in which seven of the eight cortone organs are clearly visible. The five lateral organs that together form the penasco lipid organ, a single lateral organ and one of two ventral cortone organs are evident. The neurons of the cortone organ are labeled with the neuronal marker, a monoclonal antibody 22 C 10, and shown in red.
The ligament cap and attachment cells are labeled with Antifa tubulin 85 E and are shown in blue. The cap and ligament cells are additionally labeled with a corti tonal organ. Specific GFP reporter harboring a regulatory element for the Delilah locus.
Here, the wing corti tonal organs at the ventral radial vein are shown in this confocal micrograph of a 35 hour old pupa. The neurons are marked with the neuronal marker 22 C 10, and shown in red and in panel B.The ligament and cap cells are co labeled with Antifa tubulin 85 E antibodies shown in blue and in panel C, as well as a corti tonal organ. Specific GFP reporter transgene shown in green.
After watching this video, you should have a good understanding of how to visualize proprioceptive cortone organs in both larva and pupa in oph. While attempting this procedure, it is important to remember that it was optimized for the use of the described antibodies. Different antibodies may require slightly different fixation and staining conditions.
Thus, when using this procedure with different antibodies, one should find the optimal conditions for the antibodies in use.
