This protocol is significant because this is an efficient method for the isolation of Drosophila pupal eye tissue for multiple downstream applications. The main advantage of this technique is that it allows the quick dissection of a large number of pupal eyes without damaging the tissue. Dissection of the Drosophila pupal retina will be challenging for novices.
Practicing the technique is the best way to improve the speed of the dissections and the quality of results. After placing the Drosophila pupae onto a black dissecting dish, lay a fresh piece of double-sided tape onto the dissecting dish, away from the pupae, and use a pair of forceps to carefully place the pupae dorsal side up onto the tape. Place the dish under a stereo microscope, and use forceps to remove the operculum of each pupa.
Use microdissection scissors to slice each pupal case, allowing it to be flapped open to reveal the head, thorax, and anterior abdominal segment, and secure the edges of the pupal case to the double-sided tape. Pierce the abdomen of each pupa with sharp forceps, and remove the pupa from its pupal case. Place the pupa on the dissection dish, away from the tape, and cover the pupa with 400 microliters of ice-cold PBS.
Use forceps to grasp each one by the abdomen, and use the microdissection scissors to make one clean cross-sectional incision through the thorax, cutting the pupae in half. Using two pairs of fine forceps, grasp the cut edges of each thorax epithelium and gradually tear open the thorax and head capsule, exposing the eye-brain complex and the surrounding fat tissue. Then, without grasping the tissue, use forceps to guide each translucent, off-white, dumbbell-shaped eye-brain complex, away from the remnants of the head capsule.
After dissecting six to 10 pupae, cut a micropipette tip with a clean razor blade to a diameter of about one millimeter, and lubricate the tip with a mixture of PBS and fat. Use the lubricated pipette tip to transfer the eye-brain complexes to one well of a nine-well glass dish containing 400 microliters of PBS on ice, and then transfer the tissue in 20 microliters of PBS into at least 250 microliters of fixative for a 35-minute incubation on ice. At the end of the fixation, use the same pipette tip to transfer the tissue into a third well containing 400 microliters of fresh PBS for five to 10 minutes on ice.
To block any non-specific antibody binding, transfer the eye-brain complexes into 400 microliters of PBS plus Triton X for a 10-to 60-minute incubation on ice. At the end of the incubation, aliquot 10 microliters of the primary antibody solution into the necessary number of wells of a 72-well microwell plate, and use a P10 air-displacement micropipette with a modified 0.5-millimeter tip lubricated with PBS plus Triton X to transfer no more than five eye-brain complexes and no more than three microliters of PBS into each well of antibody solution. After an overnight incubation at four degrees Celsius, wash the samples two times in 400 microliters of PBS plus Triton X in one well of a nine-well glass dish for five to 10 minutes per wash.
After the third wash, transfer the tissue into 100 microliters of an appropriate secondary antibody solution for two hours at room temperature, protected from light, following by three five-to 10-minute washes in 400 microliters of PBS plus Triton X and one wash in PBS. Then, equilibrate the samples in 200 microliters of mounting medium for one to two hours. At the end of the incubation, transfer the samples to a microscope slide, and use a sturdy tungsten needle to pin an eye-brain complex to the slide under a dissecting microscope.
Then, use a fine tungsten needle to slice each eye away from its associated optic lobe and to maneuver each eye to the surface of the microscope slide so that the basal surface of the eye is adjacent to the slide. Then, apply a clean coverslip over the samples, and seal the coverslip with nail polish. To prepare pupal eye tissue for Western blot analysis, first dissect 10 to 16 pupae as previously demonstrated in two batches, 10 to 15 minutes apart in PBS supplemented with protease and phosphatase inhibitors.
After briefly rinsing the isolated eye-brain complexes two times in individual wells of a nine-well glass dish on ice in 400 microliters of PBS plus inhibitors, transfer all of the eye-brain complexes into 400 microliters of ice-cold PBS plus inhibitors decanted onto a clean black dissecting dish. Under a clean dissecting microscope, use a sturdy tungsten needle and a fine razor blade to cleanly cut each eye from the optic lobe, and use a lubricated tip to transfer the eye-brain complexes into five microliters of PBS plus inhibitors into a 500-microliter microcentrifuge tube on ice. Then, add five microliters of Western tissue lysis buffer and 10 microliters of 2X concentrated protein sample buffer to the tube.
To process the eye-brain complexes for RNA extraction, wearing gloves, clean the benchtop, microscope, dissecting tools, and black dissecting dishes with 70%ethanol and RNase decontamination reagent. After allowing the area to dry, dissect a total of 30 to 40 pupae in batches of six to eight. After each batch of eye-brain complexes have been dissected, transfer the eye-brain complexes from the nine-well dish into 400 microliters of fresh, ice-cold, nuclease-free PBS on a clean dissecting dish, and use a sturdy tungsten needle and a fine razor blade to cleanly cut each eye from the optic lobe.
When all of the eyes for a batch have been removed, transfer them into a sterile, RNase-free microcentrifuge tube, and flash-freeze the samples in liquid nitrogen before proceeding to the next batch. The pupal eye is an easily accessible tissue that serves as an excellent model for investigating developmental processes, including those that drive morphogenesis. Around 40 hours after puparium formation, the final arrangement of cells is achieved.
This is an ideal age at which to assess the consequences of a genetic mutation or modified gene expression. For example, following ectopic expression of Diap1, a core inhibitor of apoptosis, the consequent increase in the number of lattice cells can be compared to that observed in a control retina. Apoptosis can also be assessed more directly by utilizing antibodies to activate a death caspase-1 or by using other approaches for detecting dying cells.
Western blot analyses of eye lysates can be used to determine the presence or relative expression of proteins of interest or to compare protein expression at different developmental time points. It is important to note that dissecting more than 60 eyes per genotype or experimental condition is optimal for isolating sufficient high-quality RNA. The most important thing to remember is that the pupal retina is extremely fragile, and great care must be taken during its dissection to ensure its integrity.
