The overall goal of this procedure is to visualize isolated ovaries from late third instar drosophila larvae by confocal microscope. This is accomplished by first selecting female larvae from a synchronized population. The second step of the procedure is to dissect out an intact fat body, separate it from the intestine and cuticle, and place it on ice.
The third step of the procedure is fix stain and wash fat body with embedded ovaries. The final step of the procedure is separating ovaries from fat body while mounting. Ultimately, results can be obtained that show cell specific staining through immunofluorescence microscopy.
Generally, individuals new to this method will struggle because the fine dissections and mounting require a great deal of practice. Five days before dissection, add yeast to a fresh 25 millimeter vial of fly food. Place seven to 16 mated female fruit flies into the vial.
Allow the females to lay eggs for two to four hours or until there are about 30 eggs per vial. Incubate the egg containing vials at 25 degrees Celsius with a minimum of 70%humidity. After five days, the larvae have reached the late third instar.
Fill a nine well glass dissecting dish with ringer's medium. Next, prepare specially made molds fitted with a nylon mesh in the Petri dish containing ringer's.Medium. Place the dish on ice using fine forceps.
Pick 10 to 15 larvae from the vial walls and place them in the dissecting dish. Place the dissecting dish under a microscope for female selection and dissection. Female larvae can be distinguished from males by their goads.
Male testes are easily identified as big, clear ovals embedded in the posterior third of the fat body. Female ovaries located at the same part of the fat body can be identified as much smaller, clear round spheres. If the ovaries are not visible, the female larvae are identified by the absence of testes.
Transfer a female larvae to a clean well To begin the dissection, use forceps to hold the larvae down just posterior to the brain. Remove the head with a second pair of forceps. Place the remaining posterior part of the larvae on the dorsal side with the trachea facing down.
Hold the larvae by the posterior sphericals with one pair of forceps. Then push the forceps inward slowly at the same time, use the other pair of forceps to slide the cuticle posteriorly until about half of the larval fat body emerges firmly. Hold the posterior end of the cuticle with one pair of forceps and loosely hold the rest of the cuticle with the other pair gently and slowly pull away the posterior end so that the cuticle and the attached intestines slide through the gap.
At the end of this process, disconnect the intestine and the cuticle from the anterior part of the fat body. Wet a pasture pipette thoroughly with ringers medium from a well containing dissected larvae. Use the pasture pipette to transfer the fat body into the ice cold ringers medium in the cell strainer.
The next step is to fix and stain the preparation at 5%formaldehyde in ringer's medium and incubate the fat body for 20 minutes with gentle agitation on an orbital shaker. Unless specified all steps are performed at room temperature. Wash the fat body three times with 1%PBT the first time for five minutes, the second time for 10 minutes, and the third time for 45 minutes.
Block with 0.3%PBTB for one hour with gentle agitation. Following the agitation. Transfer the fat body to a 0.2 milliliter tube.
Remove the excess liquid and add the desired first antibody diluted in 0.3%PBTB and incubate overnight at four degrees Celsius with gentle agitation on a roller. The next day, transfer the fat body back to the mold. Next, wash the fat body three times for 30 minutes each with 0.3%PBTB with gentle agitation.
Then block with 0.3%PBTB supplemented with 5%normal donkey serum for one hour. With gentle agitation, transfer the fat body to a 0.2 milliliter tube and add an appropriate secondary fluorescent antibody diluted in the blocking solution. Place the tube in a rack and place the rack on a roller incubate for two hours with gentle agitation.
If the antibody is fluorescent, incubate in the dark from this point onward. After two hours, transfer the fat body back to the mold and wash three times for 30 minutes each with 0.3%PBT with gentle agitation to dissect out and mount the ovaries. Transfer the fat body to a clean einor tube very carefully.
Remove all liquids with a pasture pipette and immediately cover with vector shield Mounting media. Cut the end of a pipette tip and carefully suction up the fat body with minimal volume of mounting media. Transfer the fat body onto a microscope slide.
The next step is to use two nickel plated pin holders holding 0.1 millimeter diameter pins to spread out the fat body under a stereo microscope, localize the ovaries by looking in the posterior third of the fat body. The fat body has a flower shape where it surrounds the ovaries. Dissect out the flowers from the rest of the fat body and discard the ladder.
Remove the fat body surrounding the gonad by crossing the two pins carefully around it and teasing it away from the ovary. Place the isolated gonad on the slide away from the fat body residue and discard the fat body residue. Cover the slide with the cover.
Slip and seal with nail polish. Visualize the ovary directly using a confocal microscope. These figures are from two ll three ovaries stained with different combinations of antibodies.
In the first image, one antibody outlines the somatic cells and stains. The fuso fuso indicated by the arrows are intracellular organelles. Within the primordial germ cells, shown in green are the terminal filament and cap cells, which together form the somatic cells of the niche, the arrowhead point to the cap cells.
This figure shows the intermingled cells in magenta, which directly contact the germ cells depicted in blue. The somatic cells of the ovary are outlined in green After its development. This technique paved the way for researchers in the field of stem cell biology to explore niche formation and stem cell establishment in the Tezo ovary.
