Larval Ring Gland Dissection: Isolation of Developmental Endocrine Organs from Drosophila

- To dissect the ring gland, you will need a dissecting microscope, two forceps, a fine gauge needle to serve as a dissecting knife, and a buffered solution. Find the ring gland by identifying the head of the larvae, which contains the mouth hooks. Travel along the length of the larvae to find the brain, which is between the thorax and the abdominal sections. The ring gland is smaller than the brain and is situated on top.

The larval ring gland is a complex endocrine structure that contains three different endocrine organs. The corpus allatum sits in the center of the ring gland and produces juvenile hormone, which regulates development and aging. The prothoracic gland surrounds the corpus allatum and makes up the majority of the structure. Ecdysone, a hormone involved in insect malting, is made by this gland. Below the prothoracic gland is the corpus cardiacum, which produces adipokinetic hormone, a nutrient regulator.

In this example, we will dissect larval brains and ring glands for immunohistochemistry.

- 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. After immunostaining the larvae, use a disposable pipette 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 disks with a needle knife.

- It is quite essential to use fine forceps with sharp edges during dissection. I also strongly recommend that you sharpen forceps with an Arkansas grinding stone to bring their edges together without any gaps.

- To transfer the samples, use a micropipette 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 coverslip over the reagent, and then over the samples.