We describe a protocol to dissect, immunostain and mount larval and adult Drosophila brains with a special emphasis on the optic lobes. Given the optic lobe's complex three-dimensional organization, it is anticipated that the protocol described here will provide researchers with a greater understanding of the relationship between mounting orientation and the optic lobe structures visualized. This protocol describes three mounting strategies for both larval and adult optic lobes, each of which provide an optimal angle to visualize a specific optic lobe structure during imaging.
Before beginning the dissection, fill all wells of a three well plate with 400 microliters of PBS per well, and use forceps to gently select 15 wandering third instar larvae crawling on the inside of a drosophila vial or bottle. Place all larvae in the first well of the three well plate, and transfer one larva into the middle well of the plate. Using the non-dominant hand, restrain the body of the larva at the base of the well and use the dominant hand to gently grasp the larva mouth hook.
Pull the mouth hook away from the body to remove the brain. Subsequently, remove the accessory tissues, including the imaginal discs. Then place the brain into the third well of the plate.
When all brains have been dissected, use a P200 pipette to carefully remove the PBS from the third well, leaving just enough solution to keep the brain submerged. Add 500 microliters of freshly prepared cold fixation solution to the well. Gently stir the solution with the forceps so that the brain swirl in the dish, and cover the well with a glass microscope slide.
Then place the dish on ice for 30 minutes to fix the tissue. Wash the brains five times with 400 microliters of fresh PBS plus triton per wash. The brains are now ready for primary antibody incubation.
For adult brain dissections, anesthetize 10 to 15 adult flies and place them on a lab wipe over ice. Use forceps to gently transfer one adult fly by the wing into one well of a three well dissection plate containing 400 microliters of PBS. Use a pair of forceps in the non-dominant hand to hold the thorax of the fly against the base of the well, and use ultra fine forceps with the dominant hand to gently pull the head from the rest of the body.
Discard the body onto a lab wipe with the non-dominant hand and use the dominant hand to hold the head against the base of the well by the proboscis. Use both forceps to peel away each region of the cuticle between the eyes, until the brain is exposed, and remove any accessory tissues that remain attached to the brain. Place the cleaned brain into the third well and dissect the next brain as demonstrated, until 15 brains have been obtained, or a maximum dissection period of 30 minutes has elapsed.
Fix the brains with 500 microliters of fixation solution for 20 minutes at room temperature, and wash the brains five times with PBS plus triton, as demonstrated. Adult brains are now ready for primary antibody incubation. Replace the last wash with two drops of fluorescent safe mounting medium to submerge the brains, and add a single drop of mounting medium to the center of a new glass microscope slide.
Use forceps to grasp each larval brain by the ventral nerve cord for transfer onto the slide. To visualize progenitors and neurons from the central region of the outer proliferation center, Lamina or lobula plug, ensure that the ventral nerve cord projects out over the lobes, place the larval brains onto the slide with the anterior side of the tissue facing up. To visualize cells belonging to the tips of the outer or inner proliferation centers, ensure that the ventral nerve cord projects out from under the lobes.
Place the larval brains onto the slide with the posterior side of the tissue facing up. To visualize the crescent of neurons from the inner and outer proliferation centers in both the dorsal ventral and medial lateral axis, use a pair of sharp forceps or a tungsten needle to split the brain lobes down through the ventral nerve cord, starting from the cleavage point between the lobes. Reorient each lobe with the lateral side facing up.
When the larval brains have been appropriately oriented, place a small drop of mounting media on either side of the brains. Place one cover slip onto each drop, and place a final cover slip over the brains so that the right and left edges rest on the other two cover slips, forming a cover slip bridge. Then use nail polish to seal the edges of the cover slip bridge to secure the mounted brains.
After the last secondary antibody wash, perform a final wash with 400 microliters of PBS. Replace the PBS with three drops of fluorescent safe mounting medium to submerge the brains. Add a drop of mounting media to the left and right thirds of a glass microscope slide.
Place a cover slip over each drop to build a cover slip bridge. Seal the outer edges of the right cover slip with nail polish and add a single drop of mounting media between the cover slips. Using forceps, gently transfer the brains to the slide with extra caution to prevent damaging the optic lobes.
To visualize the cell bodies of the lamina and medulla neurons, use a tungsten needle to orient the brains in an anterior position with the antenna lobes protruding outward. To visualize neurons of the lobula complex, orient the brains in a posterior with the antennal lobes facing down against the slide position. To visualize all optic lobe neuropils in a single plane, orient the brains in a horizontal position, line up the brains in an anterior mount, then use a tungsten needle to rotate each brain 90 degrees upwards so that it sits on its ventral side, resting on the edge of the cover slip.
When the adult brains have been appropriately oriented, place a final cover slip over the brains such that the right and left edges overlap with the other two cover slips to form a bridge, seal the slide with nail polish. Here is an image of a larval optic lobe in the anterior mounting orientation. In the anterior mounting orientation, the epithelium of the central louder proliferation center, medulla neuroblast and lamina neurons appear at the surface as bands of cells that wrap around the brain.
Deeper into the brain, in the middle of the Z-stack, the main outer proliferation center epithelium and its respective neuroblast and neurons are visible. Additionally, the epithelium of the inner proliferation center and neurons of the lobula plug are visible in these intermediate slices. The deepest Z slices depict the other side of the brain where the posterior tips of the outer proliferation center are located.
The superficial tip of the inner proliferation center is also present. Note that these would be the first structures visible if the optic lobe was mounted posteriorly. Here is an image from an optic lobe mounted on its side.
At the surface of the lateral mount the lamina crescent is visible and the lobula plug crescent is located between its arms. The medulla neuronal crescent appears at a slightly deeper Z position. Here is an image of an adult optic lobe mounted in an anterior orientation.
Since the medulla is located at the surface of the anterior mount, the medulla cortex is immediately visible. Cell bodies in the cortex project their arborizations into the neuropil, which can be visualized at an intermediate Z position. The lobula also appears at this level, oriented perpendicular to the medulla.
At the deepest Z position, the final neuropil of the optic lobe, the lobula plate is visible. Note that the lobula plate would be the first structure visible in a posterior mount. Here is an image of an adult optic lobe mounted in a horizontal orientation.
When the brain is flipped 90 degrees on its side to a horizontal position, all of the neuropils and cortices of the optic lobe are visible in a single plane. When performing this protocol, the most important thing to remember is to keep the brain tissue submerged in solution throughout the protocol. If the brains are exposed to air, the quality of the stain and, therefore, the final images will be significantly reduced.
An understanding of how mounting orientation affects optic lobe visualization is important for live imaging. Larval and adult brains can be cultured and imaged over several days to follow cell divisions or changes in your own morphology. Here, the mounting orientation is critical, as the cell types of interest must be close to the cover slip for optimal detection of the fluorescent signal in vivo.
