This protocol can be used to study wound-induced polyploidization. A conserve tissue repair process, in which cells grow instead of divide in the adult fruit fly. A simple puncture wound can be made to induce polyploidy in the fly epithelium within just two days.
Epithelial cell size, ploidy and organization, can then be assessed easily. Begin by collecting two vials, containing 10 to 15 newly closed female fruit flies of the strain of interest. And aging the flies in fresh food vials, with approximately five male flies per vial at 25 degrees celsius, until three to five days old.
For abdominal wounding, at the end of the incubation, use a single 0.1 millimeter stainless steel pin to assemble several pin holders, with the sharp end of each pin pacing out. Anesthetize the aged female fruit flies on a carbon dioxide fly pad under a stereo microscope. And use a paintbrush to arrange the flies into a row.
Wearing safety glasses, with a pin holder in one hand and forceps in the other, use the forceps to position the flies with their ventral abdomens facing up. Then puncture the adult female flies within the epithelial blue right region of target A4 on either side of the ventral midline stir nights and return the wounded flies to the food vial until the desired experimental time point post-injury. At the experimental endpoint, check for the presence of the wound and scar in each anesthetized fly under the stereo microscope and fill one well of a nine well glass dissection dish with Grace's solution.
Using a pair of forceps, grasp a wounded female fly by the dorsal side of the thorax and submerge the fly in the well of solution. With the other hand, use a second pair of forceps to puncture and remove the dorsal cuticle below target A6, at the rear of the fruit fly. If the internal organs are not extracted, apply pressure to the dorsal side of the abdomen to squeeze out the remaining organs.
Use the forceps to snap off the full abdomen at the thorax junction above target A2 and transfer the abdomen to a well containing approximately 100 microliters of Grace's solution. When all of the abdomens have been collected, reduce the volume of Grace's solution in the collection well to 30 microliters. Use forceps in one hand to hold the abdomen's dorsal side down and use the other hand to insert the bottom blade of Venice spring scissors into the abdominal cavity of each abdomen and cut along the dorsal midline until the abdomens are fully open.
Add 30 microliters of Grace's solution into each mounting area of a dry dissecting plate with 4.1 millimeter pins per abdominal mounting area And place the filleted abdomen into the droplet of solution. When all of the abdomens have been filleted and placed, pin the abdomens to the dish on the four dorsal corners. Taking care that the tissues are lying flat without tearing or overstretching the abdominal tissue.
Then replace the Grace's solution with 30 microliters a fixative solution per mounting area. And place a tape label on the bottom of each dish to mark each control and experimental group. To mount the fly tissue samples.
After staining, use forceps to unpin the abdomens from the dissecting plate under the stereo microscope. And use forceps to transfer each sample by its dorsal flank into approximately 30 microliters of mounting medium on individual glass cover slips. Under the stereo microscope orient the abdominal tissue so that the inside is facing down toward the cover slip and use forceps to pull the oriented abdomens to the edge of each media droplet.
Place each mounted cover slip onto a labeled glass slide. And use a lab tissue to remove any excess mounting medium. Then seal the edges of the cover slip with clear nail Polish and store the slides in the slide box at four degrees celsius until their imaging.
The septate junction protein fast three which labels cell cell junctions, provides an indicator of whether any processing perturbations occurred during the preparation. Abdomens with large scratches of unstained area that perturb the wound area, must be discarded and not included in the analysis. Wound repair is complete when a central large multinucleated cell covers the wound scab.
Gaps of greater than 10 micrometres on the epithelial sheet are considered defects in wound closure and re-epithelialization. In this representative analysis using flies with inhibited mitotic cell activation, 52%of the wounds were unable to form a continuous epithelial sheet over the wound scab. This membrane wound healing essay provides more information to the extent of the wound repair defect allowing re-epithelialization defects to be grouped as either completely open, partially closed or completely closed.
For example, in this experiment the inhibition of wound induced polyploidization by the simultaneous blocking of endo replication and cell fusion, caused at 92%of the epithelial wounds to remain completely open. While activation of the mitotic cell cycle resulted in an epithelial wound closure defect. In addition, cell cycle activity was detected by incorporation of the thymidine analog EDU and epithelial ploidy was determined by directly measuring the nuclear DNA content.
When dissecting out the abdominal epithelium, take care to not touch the tissue with any of the disecting tools as this will scratch the sample. After dissection, cell proliferation, cell death, cell loss incision size or nuclear DNA content assessments can be performed to better understand wound induced polyploidization. or other processes within the epithelium.
