- To orient a larva for fillet dissection, place it in a silicone-coated dish containing cold saline and identify its dorsal and ventral sides. Tracheal tubes running along its length mark the dorsal side, while abdominal denticle belts identify the ventral side.
Then, with the ventral side up, gently stretch the larva and pin each end to the bottom of the dish. This position will keep the dorsal clusters of sensory multi-dendritic neurons and the associated epidermal cells intact as the dissection proceeds.
Use dissecting scissors to cut along the ventral midline of the larva. Pin the cuticle to the plate to flatten the preparation and remove internal structures to reveal the underlying muscle tissue. Then, carefully remove the appropriate muscle to expose the epidermis and gain access to previously obscured neurons while preserving their dendritic morphology.
In the example protocol, we will see a detailed demonstration of larval fillet preparation and muscle removal to view sensory da neurons and epidermal cells.
- To begin, add enough cold saline to a silicone elastomer dish to cover the bottom surface. Add the subject larva to the dish and place it under the stereo microscope, ensuring proper placement of light. Position the larva with the ventral side up, which can be identified by the abdominal denticle belts.
Stretch the larva in the anterior-posterior direction, and using insect pins, pin the head and tail to the bottom of the dish. With a pair of fine dissecting scissors, cut along the ventral midline of the larva from one end to the other. Spread the larva open and pin the cuticle tissue at the four corners so that it lies flat.
Using forceps, remove the internal organs, including the CNS, gut, and trachea. Adjust the insect pins so that the fillet is taut, but not maximally stretched.
Locate the dorsal midline of the larva, where muscle tissue is absent and then position a forceps prong at the interior of this boundary. Slide the prong between the muscle and epidermis, taking care to minimize contact with the epidermis.
- To minimize damage to the epidermis, dissect with the flattest possible prongs. And try dissecting in calcium-containing saline. That could help create space between the muscle and the epidermis.
- Next, pull the forceps upwards to break the attachment of the muscle to the body wall at one anchor point. Repeat this process for the remaining muscle segments of interest.
Readjust the insect pens to maximally stretch the larval fillet in all directions.