Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

The overall goal of this procedure is to visualize the steroidogenic organs and their interactive organs in larvae or adult females of the fruit fly, Drosophila melanogaster. This method can help understand insect steroid hormone biosynthesis, and the neuronal regulatory mechanism underlying mating and metamorphosis. The main advantage of this technique is that the innervation of steroidogenic organs and the relative portion of their interactive organs remains intact.

Generally, individuals new to this method will struggle because the steroidogenic organs in fly larvae are quite tiny and transparent. My lab and I are pleased to share our dissection protocols. So, let's get started.

After preparing the larvae, start the course dissection in a three centimeter dish filled with PBS, under a dissecting microscope. First, grip the mouth hook with forceps. Then, using micro scissors, sever the body at about a third of the length from the anterior tip.

Next, hold the anterior length with one pair of forceps and use a second pair to gently push the tip of the head into the body, to ultimately turn the larval body inside out. Prepare up to 20 larvae in this manner within 10 minutes, and then transfer them into fixative solution. After 30 minutes, wash the preparations, and proceed with immunostaining.

To begin, leave the larvae immersed in water for about one hour, to asphyxiate and thus immobilize the larvae. Then, place a larva dorsal side up in a drop of PBS on a silicone-coated dish. The dorsal side has the two tracheal tubes.

Under a dissecting microscope, use forceps to insert an insect pin into the anterior tip. Then, stretch the body to the posterior side and put a second pin into the posterior tip. Next, make a small incision near the tail using micro scissors.

From the incision, carefully cut the dorsal cuticle longitudinally along the dorsal midline, toward the head, while not damaging any of the tissues under the cuticle. After making the incision, stretch the body walls apart, and secure them with insect pins at the corners. Then, using forceps, remove the anterior fat body and salivary glands, thus exposing the brain-ring gland complex on the surface.

Now, aspirate the PBS, and immerse the preparation in fixative solution. After 30 minutes, use forceps to remove the insect pins and transfer the preparation into a microtube filled with 0.3%PBT. Then, proceed with immunostaining.

After immunostaining the larvae, use a disposable pipet to transfer the samples to a dish filled with 0.3%PBT. Under a dissecting microscope, grip the cuticle or mouth hook with one pair of forceps. Then, use a 27 gauge needle attached to a one milliliter syringe, like a knife, to remove the brain-ring gland eye disc complex from the body cuticle, by cutting away the anterior tip of the esophagus and eye discs.

Next, using forceps, carefully separate the esophagus and gut from the brain-ring gland eye disc complex. Pull the gut out to the posterior side, since the esophagus passes through the brain above the ventral nerve cord. Finally, to isolate the brain-ring gland complex, carefully cut the connections between the brain disc, eye discs, and leg discs, with a needle knife.

It is quite essential to use fine forceps with sharp edges during dissection. I'd strongly recommend that you sharpen forceps with a piece of grinding stone to bring their edges together without any gaps. To transfer the samples, use a micro pipet with a wide bore tip.

Deposit the samples onto the center of the glass slide. Then, use forceps to position the samples onto their ventral sides, so the ring gland, which is located on the dorsal side of the brain, can be imaged. Next, tilt the slide and wipe off as much of the excess PBT as possible.

Then, put a drop of mounting reagent near the samples and use forceps to slowly lower a cover slip over the reagent, and then over the samples. To dissect the adult female ovary, anesthetize flies with carbon dioxide gas. Then, cut off their heads and transfer their bodies to a three centimeter dish filled with PBS.

Next, under a dissecting microscope, grip the dorsal side of the thorax with forceps, and take hold of abdominal segment A-5 or A-6 with a second pair of forceps. Then, peel away the abdominal cuticle towards the posterior side. Before proceeding, clean the sticky cuticle off the forceps.

Next, pinch an ovaduct, and isolate the ovary. Then comb out and spread the tips of the ovary using forceps. Once spread, immerse the ovary in fixative solution for 30 minutes, to prepare them for staining.

To make a preparation that maintains the innervation of the ovary, transfer anesthetized female flies to a silicone-coated dish filled with PBS. Under the dissection microscope, hold the proboscis, and peel away the head cuticle to expose the brain. From the brain, remove the attached trachea, leaving the brain connected to the ventral nerve cord.

Then, hold the thorax by the dorsal side, and remove the legs and wings. Next, peel away the ventral cuticle of the thorax, beginning at the base of each leg. Once the VNC is exposed, very carefully remove the residual dorsal cuticle and muscles attached to the VNC, using forceps.

Do not damage the connections between the brain and the VNC. Next, using forceps, peel away the abdominal cuticle to expose the ovaries and their interactive organs, which includes the prop, the gut, the ovaduct, the uterus, and the accessory gland. Finally, remove any residual tissues, including the trachea, and the fat body.

Then, immerse the samples in fixative solution for 30 minutes, to prepare them for immunostaining. After immunostaining, transfer the samples to a glass slide with 0.1%PBT using a micro pipet. Then, view the slide with a microscope, and separate the strings of the ovarioles from each other using forceps.

Do not injure the tips of the ovarioles during this process. They contain the germaria. When preparing the brain to reproductive organ complex, remove any residual cuticle, and spread the structures out onto the slide.

Next, wipe off most of the excess PBT, and then apply a drop of mounting reagent to the sample. Next, slowly place a cover slip onto the slide using forceps and store the slide at four degrees celsius in the dark. Later, observe the samples under the microscope, and bring the steroidogenic organs into the field of view.

Once the view is fixed, switch the imaging system to the image acquisition mode. During the larval stages, the prothoracic gland was labeled with antibodies against ecdysteroidogenic enzymes, including the anti-shroud antibody. To visualize the neuronal connection between the prothoracic gland and the brain, the brain-ring gland complex was dissected.

The filet dissection method shows the stomatogastric nervous system, in which a group of serotonergic neurons project to the prothoracic gland, the proventriculus, and the pharyngeal muscles. In adult females, ovarian ecdysteroid affects many aspects of ovogenesis, such as GSC proliferation. The GSCs are located in the germarium at the tip of each ovariole in the ovary.

Within the germarium, the GSCs were specifically labeled using two antibodies, 1B1, and DE-cadherin. To visualize the innervation of the ovary, the ovary was dissected, along with the brain, VNC, gut, prop, uterus, and spermatheca. Neurons were visualized by mCD8:GFP under the control of the n-Synaptobrevin GAL4 driver.

Additionally, muscles'structure were stained using diconjugated phalloidin. Although dissection of the steroidogenic organs can at first feel difficult, use this movie to learn the cutting point of the tissues to more easily isolate the organ without injury. We hope that our protocol is informative, not only for beginners, but also experienced researchers.

Once mastered, you can apply this procedure to your research, and brush up to fit with your needs. We hope this protocol will help the comprehensive understanding of steroid hormone biosynthesis. More detailed step-by-step protocols are available in the manuscript.

Please check it out.