The overall goal of this procedure is to dissect and stain drosophila pupal ovaries. This method can help answer key questions in development of biology, such as those pertaining to stem cell differentiation and tissue development in the model organism drosophila. The main advantage of this technique is that customizable tools are used to accommodate the small size and translucent nature of the pupal ovaries, which are otherwise difficult to isolate.

We had the idea to use his method when we conducted time course experiments, to track stem cell division in the ovary during pupal drosophila development. To begin the protocol, combine 10 male and 15 female adult drosophila flies, in a vial of normal rich fly food supplemented with yeast. Allow the eggs to develop into larvae at room temperature for three to four days.

After three to four days, use a moist fine brush with soft bristles to make a rolling movement with the brush along the wall of the vial, to transfer wandering third instar larvae, from the vial to a glass well, filled to the brim with 1X phosphate buffered saline or PBS. Wash the food debris off the larvae, by transferring them to another well filled with 1X PBS. Separate the male and female larvae with forceps.

Identify the male larvae by a pair of large round and translucent testes embedded in the fat body on the lateral side two-thirds down from its anterior end. Then collect at least 10 females from the well and place the female larvae into a separate well, filled with 1X PBS. Use forceps to transfer the female larvae gently into a new vial of fresh fly food supplemented with yeast.

Place the vial with the female larvae in a dark location to facilitate pupariation. Throughout the day, monitor the larvae for pupariation. As each larva lobalizes and develops into a pre-pupa, circle the pre-pupa against the vial and record the approximate time when it first forms into a pre-pupa.

Melt the glass tip of a pester pipette over a Bunsen burner. As the glass melts, use forceps to pull the tip horizontally away from the rest of the pipette to form a thinner tip. After cooling, break off a small portion of the pipette tip to form a neat circular opening.

Attach a bulb to the other end of the pipette and load the pipette with 1X PBS. To harvest the pupae glued to the wall of the vial apply a small drop of water along the contact zone between pupa and vial. After allowing the protein glue to dissolve for one to two minutes, gently lift the pupa off the wall with a moist fine brush.

Transfer a single pupa per well filled with 1X PBS. While grasping the posterior end of the pupa with one pair of forceps, carefully tear the anterior portion of the pupal case with another pair until the head of the pupa is visible. Grip the anterior most tip of the pupal head with forceps, and gently pull the pupa out of its pupal case.

Next separate and discard the anterior half of the pupa from the posterior half until only the abdominal sac remains. Grasp the abdominal sac against the bottom of the well with forceps in one hand, while holding the thin glass pipette filled with 1X PBS in another hand. Aim the thin pipette tip towards the opening of the sac and slowly pipette 1X PBS into the abdomen to wash away the fat body cells surrounding the pupal ovaries.

Wash away the fat body cells until at least two thirds of the fat body is gone, or until the ovaries are visible near the opening of the abdominal sac. If the ovaries are not visible inside the well, they should've remained in the abdomen. If the ovaries have come out, place a new pupa in the well and repeat this step.

Transfer the abdomen into a clear covered glass chamber filled with fixation buffer, and place the lid on top. Dissect more ovaries than needed to ensure enough ovaries would stand at the staining and mounting process. Throughout the staining process, use forceps to gently push down any floating ovaries to the bottom of the covered glass well to ensure that the ovaries are completely immersed in antibody solution.

Place strips of double-sided tape on to the flat surface of the new titer and then place the chambered cover glass containing the ovaries and fixation buffer on the adhesive surface. Incubate the ovaries in fixation buffer for 15 minutes at room temperature. Next, rinse the ovaries three times in 1X PBS with 1%Triton X-100 or 1%PBST for five, 10, and 45 minutes respectively, to allow thorough permeabilization.

Lock the ovaries in 10%normal goat serum in 0.5%PBST for 30 minutes. Then incubate the ovaries overnight in 600 microliters of primary antibody in 0.5%PBST at 4%Celsius. The next day rinse the ovaries three times for five minutes each in 0.5%PBST.

Incubate the ovaries for two hours in 600 microliters of secondary antibody diluted in 0.5%PBST at room temperature. After incubation, rinse the ovaries twice for five minutes in 0.5%PBST and once for five minutes in 1X PBS. Place a small drop of 1X PBS on to a microscope slide.

Use forceps to transfer the abdominal sac from the chambered cover glass to a glass well filled with 1x PBS. Tear apart the abdominal sac and any remaining fat body with forceps. Transfer the ovaries from the well into a drop of 1x PBS on the microscope slide by grasping the center of the ovaries between the tips of the forceps.

Add a few drops of 1X PBS to the slide when the solution dries. Once all ovaries have been dissected and transferred to the microscope slide, pipette 40 microliters of mounting medium on to a 22 x 22 millimeter cover slip and place the cover slip gently on top of the ovaries. Finally, let the slide dry overnight prior to imaging.

Immunohistochemistry specific to cell proliferation such as phospho histone H3 staining can be used to study cell division patterns of stem cells and other mitotically active cell types. Pupal stalk cells derived from swarm cells can be outlined with Fasciclin III staining. While attempting this procedure, it is best to harvest and dissect more PB than needed for the experiment, give that a few ovaries may become lost during the fat body extraction or immunohistochemistry process.

Thanks for watching and good luck with the experiment.