Encyclopedia of Experiments: Biology
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Transcript
- Perform the dissection in a dish with a buffer solution and under uniform illumination. Transfer a fly, ventral side up, into the dish and keep the preparation submerged throughout the procedure. While holding the base of the proboscis, gently pull the fly head away from the body to separate the two. The brain is located at the back, or caudal side, of the fly's head. Therefore, hold on to the front, or rostral, parts of the cuticle during the dissection to avoid damaging the organ.
To isolate the brain, hold the medial edge of one eye and grasp and pull the proboscis in the opposite direction to create an opening into the head capsule. While holding the cuticle at that eye, grasp the medial edge of the cuticle from the other eye and slowly pull the head capsule straight apart to reveal the brain. If necessary, continue to clear residual cuticle, attached air sacs, or trachea. High quality preparations will preserve structures like the optic lobes, central brain, and SOG. In the example protocol, we will see a detailed demonstration of a Drosophila adult brain dissection used to study the mushroom bodies.
- To setup for the dissection, place the anesthetized flies of interest on a cold metal pad or in a Petri dish sitting on ice. Alternatively, use a fly pad that emits carbon dioxide. Next, place a small amount of PTN in the center of the dissection dish to create a bubble of PTN. Then, under a stereomicroscope, fill the field of view with the PTN bubble under uniform illumination. Now, manipulate flies so that they are belly up, and transfer one by the abdomen into the PTN. Completely submerge the animal in the PTN and perform the entire dissection submerged in PTN. With a second pair of forceps, grasp the proboscis and pull the head off the body.
- Occassionally, the proboscis will be removed but the head will remain attached to the body. If this occurs, grab the medial edge of one retina and apply lateral force to remove the head.
- Discard the abdomen and thorax. It is critical to keep the head in the PTN during this step. Connections from the central brain to VNC clearly cannot be analyzed using this method. Next, grasp the medial edge of the left retina at the edge of the central hole in the cuticle, and slowly pull the forceps directly apart from each other to prevent tearing of the optic lobe, which is the opaque structure covered by white, stringy trachea. As the retina dissociates, there will be a slight decrease in tension.
- In order to prevent the brain from tearing, it is critically important to grasp the medial edge of each eye and very slowly pull the forceps apart. Moving too quickly can result in altered brain morphology or damage to brain tissue.
- As shown here, grasp the cuticle with each pair of forceps and very slowly pull them in opposite directions. If done correctly, the cuticle should separate from the brain tissue, leaving the brain intact. Very sharp forceps are needed for this step. Next, carefully remove the surrounding cuticle piece by piece. While removing stubbornly attached cuticle, it can help to secure the brain by gripping the remaining VNC to avoid crushing the brain. Finally, use a P200 pipette to transfer the dissected brains into a well of a plate filled with PTN for fixation and immunostaining.
