Larval Fillet Preparation: A Method to Visualize Intact Sensory Neurons and Associated Epidermal Cells
Transcrição
– To orient a larva for fillet dissection, place it de 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 para 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 para cut along the ventral midline of the larva. Pin the cuticle para the plate para flatten the preparation and remove internal structures para reveal the underlying muscle tissue. Then, carefully remove the appropriate muscle para expose the epidermis and gain access para 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 para view sensory da neurons and epidermal cells.
– To begin, add enough cold saline para a silicone elastomer dish para cover the bottom surface. Add the subject larva para the dish and place it under the stereo microscope, ensuring proper placement of light. Posição the larva with the ventral side up, which can be identified by the abdominal denticle belts.
Stretch the larva de the anterior-posterior direction, and using insect pins, pin the head and tail para the bottom of the dish. With a pair of fine dissecting scissors, cut along the ventral midline of the larva from one end para 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 para minimize contact with the epidermis.
– To minimize damage para the epidermis, dissect with the flattest possible prongs. And try dissecting de calcium-containing saline. That could help create space between the muscle and the epidermis.
– Next, pull the forceps upwards para break the attachment of the muscle para the body wall at one anchor point. Repeat this process for the remaining muscle segments of interest.
Readjust the insect pens para maximally stretch the larval fillet de all directions.
