Drosophila melanogaster Ovary Dissection: A Technique for Ex Vivo Tissue Preparation

This protocol is an excerpt from Parker et al., Studying Mitochondrial Structure and Function in Drosophila Ovaries, J. Vis. Exp. (2017).

1. Preparation of Drosophila (the tools required are depicted in Figure 1A)

1. For any of the experiments described, collect Drosophila (maintained at room temperature, or 25 °C) within 5 days of eclosion and place them in a vial filled with 5 – 7 mL of Drosophila food (see Table of Materials), with no more than 25 flies in each vial; maintain a female:male ratio of 2:1.
2. Sprinkle a small amount of granulated yeast to stimulate Drosophila egg production. Perform experimental manipulation within 2 – 4 days.

2. Dissection of Drosophila Ovaries (the tools required are depicted in Figure 1A)

1. Warm insect dissecting medium (see Table of Materials) to room temperature, 25 °C. Fill three wells of an eight-well glass dissecting dish, with 200 µL of medium in each well.
2. Anesthetize Drosophila with CO₂ by placing the needle of the blow gun under the vial plug. Place them on a fly pad. Using a dissecting microscope, sort out 5 females and place them in the first well of the dissecting dish. Handle one Drosophila at a time when performing live microscopy.
3. While looking through the eyepiece of the dissection microscope, sever the thorax from the abdomen using two pairs of forceps. Using the forceps, carefully transfer the abdomens to the second well of the dish.
4. Use one pair of forceps to hold the abdomen at the posterior end, and slowly push the ovaries out (along with the other abdominal contents) with the other pair of forceps. Should this attempt fail, carefully remove the abdominal exoskeleton by inserting the forceps into the anterior end to release the ovaries.
5. Using the forceps, hold an individual ovary by the opaque posterior end (i.e., the yolk-filled, late-stage eggs) and move it carefully to the third well of the dish for teasing to process it for live microscopy or for fixing to perform immunostaining.
6. Carefully tease the protective sheath from around the ovaries by sweeping a teasing needle lightly from the posterior to the anterior end of each ovary while holding it by the posterior end with a pair of forceps.
   NOTE: To minimize damage during teasing, bend the needle tip and zoom in on each ovary by increasing the magnification of the microscope (Figure 1A). Teasing should be effective enough to break the sheath, but it should also be carefully done to preserve the integrity of the ovarioles.