Dissection of the Eye-Brain Complex from Fly Pupae: A Method to Isolate Retinal Tissue
Transcrição
– To begin, secure a fruit fly pupa dorsal, or back, side up. Remove the operculum or the anterior end of the pupa para expose the head of the animal. Continuar para open the pupa by cutting and peeling away the casing. Once freed, cover the dissected pupa with PBS para avoid desiccation. Make a clean, cross-sectional cut through the thorax and remove the posterior part of the carcass.
To expose the eye-brain complex, which includes the two retinas attached para the optic lobes, begin tearing the epithelium from the cut-open edge of the thorax and continue para open the head capsule gradually. Then, carefully guide the eye-brain complex away from the remnants of the head capsule.
In the example protocol, we will see a demonstration of a pupal eye-brain dissection used para isolate tissue for various downstream analyses.
– After placing the Drosophila pupae onto a black dissecting dish, lay a fresh piece of double-sided tape onto the dissecting dish away from the pupae and use a pair of forceps para carefully place the pupae dorsal side up onto the tape.
Place the dish under a stereo microscope and use forceps para remove the operculum of each pupa. Use microdissection scissors para slice each pupal case, allowing it para be flapped open para reveal the head, thorax, and anterior abdominal segment, and secure the edges of the pupal case para the double-sided tape.
Pierce the abdomen of each pupa with sharp forceps and remove the pupa from its pupal case. Place the pupa on the dissection dish away from the tape, and cover the pupa with 400 microliters of ice-cold PBS. Use forceps para grasp each one by the abdomen, and use the microdissection scissors para make one clean cross-sectional incision through the thorax, cutting the pupae de half.
Using two pairs of fine forceps, grasp the cut edges of each thorax epithelium and gradually tear open the thorax and head capsule, exposing the eye-brain complex de the surrounding fat tissue. Then, without grasping the tissue, use forceps para guide each translucent, off-white, dumbbell-shaped eye-brain complex away from the remnants of the head capsule.
