Dissection of the Midgut from an Adult Fly: A Method for Isolating the Digestive Tract
Transcrição
– To begin, remove the head from an anesthetized adult fly. Cover the decapitated fly with a solution containing Phosphate-Buffered Saline, or PBS, and Bovine Serum Albumin, or BSA, para avoid desiccation. Hold the fly by the thorax with forceps, and then pull the abdomen with a second pair of forceps para expose the gut. Since the crop, or the food storage sack, will be attached para the thorax, gently pull the gut away from the thorax.
Once detached, pull the crop away from the abdomen para unfold the gut. Pull off the posterior end of the fly para begin para free the gut from the abdominal cavity. Continuar tugging the gut para pull the entire tissue out of the abdomen. Once freed, remove the foregut, Malpighian tubules, hindgut, and ovaries para leave behind only the dissected midgut, the primary site of digestion within the fly.
In the example protocol, we will dissect midguts from young and old adult flies para be analyzed further via Fluorescence-Activated Cell Sorting, or FACS.
– Before beginning the dissection, anesthetize one vial of 40 flies with carbon dioxide on a standard fly bed. Then use a razor blade para decapitate all of the flies and transfer them para a dissection dish coated with agarose gel. Pour enough cold PBS plus BSA into the dish para cover the agarose. The flies will float.
Then, for each insect, de turn, grab a fly abdomen with one pair of forceps, and the thorax with another. Separate the thorax from the abdomen. The gut will be visible, and the crop will most likely still be connected para the thorax. Grab the gut and pull it out of the thorax. Then, grab the crop and pull the gut slightly anteriorly away from the abdomen para unfold it.
Next, use one of the forceps para grab the posterior end of the abdomen, and the other para grasp the edge of the anteriorly open cuticle. Pull the posterior end away para break the cuticle, and continue pulling posteriorly very gently, until the entire gut has been pulled out of the abdominal cavity. Remove the foregut, Malpighian tubes, hindgut, and ovaries, leaving the bare midgut.
As the midguts are isolated, use a glass Pasteur pipette para periodically transfer several of the intestinal tissues at a time para a 1.5-milliliter microcentrifuge tube containing cold PBS plus BSA on ice.
