The overall goal of this experiment is to label mitochondrial DNA replication in drosophila adult ovaries by EdU Staining. This method can help answer key questions in the mitochondrial field such as the process of mitochondrial biogenesis and the mitochondrial DNA inheritance during development. The main advantage of this technique is that it allows for good structure preservation and efficient fluorescent dye penetration of whole bound tissues.

To begin the experiment, take a vial with drosophila medium containing dry yeast and culture 10 adult female flies with 10 adult males at 25 degrees Celsius for two to three days. Ensure flies remain well fed throughout incubation. Anesthetize the flies on a carbon dioxide fly pad and pick female flies with a paint brush.

Add several drops of room temperature drosophila medium, supplemented with 10%FBS, to a dissecting pad, and place it under a stereo microscope. Using sharp fine nosed forceps, grab a fattened female fly at the lower thorax. Keeping all tissue immersed in medium, with a second pair of forceps, tug gently at the extreme posterior of the fly until the tissues in the abdomen are exposed.

Next, detach the two ovaries from the surrounding gut and extraneous tissue. Open the ovaries by pulling gently and expose the ovarioles. To aid penetration of the reagents, pass the tips of the forceps between each ovariole a couple of times.

Finally, transfer the ovaries to a 1.5 milliliter micro centrifuge tube containing 500 microliters of clean drosophila medium with 10%FBS. Repeat the dissection to collect as many ovaries as desired, placing 10 to 15 in each micro centrifuge tube. To begin labeling, aspirate the medium from the tubes containing the ovaries, ensuring the tissue remains immersed under the solution throughout, and replace it with 500 microliters of medium containing seven micromolar aphidicolin, a nuclear DNA synthesis blocking agent.

Incubate the ovaries for three hours at room temperature on a bench top rocker with gentle rotation. If drug treatment is desired, add the appropriate concentration of the chosen drug into the medium after two hours of incubation, and then allow the incubation to proceed for the desired time length for the selected drug. Remove the medium from the ovaries and rinse briefly with fresh drosophila medium two times.

Add one milliliter of medium containing ten micromolar EdU and seven micromolar aphidicolin and incubate the ovaries at room temperature for two hours with gentle rotation. Finally, remove the liquid containing EdU and aphidicolin and wash the ovaries with clean drosophila medium twice for three minutes each. To fix the tissue, first incubate the ovaries in 4%paraformaldehyde for twenty minutes at room temperature with gentle rotation.

Then remove the fixative and wash the tissue twice in one milliliter of 3%BSA in PBS, for five minutes each time, with rotation. For permeablization, remove the wash solution, add one milliliter of 0.5%Triton X-100 in PBS, and then incubate the ovaries at room temperature for twenty minutes with rotation. Finally, remove the solution and wash the tissue twice in one milliliter of 3%BSA in PBS.

Before the experiment, prepare and store the EdU buffer additive according to the manufacturer's instructions. Fully dissolve the EdU buffer additive powder into two milliliters of deionized water to make a 10x stock. To begin detection, prepare fresh EdU reaction buffer by diluting the 10x EdU reaction buffer with the ionized water to one milliliter of 1x working solution, which can label two tubes of ovaries.

Make 100 microliters of fresh 1x EdU additive buffer solution by diluting the 10x stock 1:10 in deionized water. Prepare one milliliter of EdU reaction cocktail by combining the reagents in the following order:860 microliters 1x EdU buffer, 40 microliters copper sulfate, 2.5 microliters dye azide, and 100 microliters EdU buffer additive. It is important to add the reagents in this specific order and use within 15 minutes of preparation.

Remove the BSA solution from the samples and add 0.5 milliliters of the EdU reaction cocktail to each tube. Incubate the samples at room temperature for 30 minutes with gentle rotation. Protect the samples from light.

Finally, wash the samples once in one milliliter of 3%BSA in PBS, and then wash once in one milliliter of PBS. To begin antibody labeling, first remove the wash solution from the ovaries and add one milliliter of blocking solution containing 0.2%BSA and 0.1%Triton X-100 in PBS. Incubate the samples for 30 minutes at room temperature with gentle agitation, taking care at each labeling step to protect the samples from light.

Remove the blocking solution. Replace it with primary antibody diluted in blocking solution, and incubate overnight at four degrees Celsius in the dark. Wash the samples with one milliliter of blocking solution three times, for ten minutes each, and then wash two further times at 30 minutes each to minimize background staining.

Finally, remove the blocking solution. Next, incubate the tissue in 500 microliters of secondary antibody diluted in blocking solution for two hours, at room temperature, with agitation. Repeat the wash steps using one milliliter of blocking solution three times for 10 minutes, and twice for 30 minutes.

Finally, rinse the tissue with one milliliter of PBS to remove the detergent. Carefully remove all of the PBS, and immediately cover the tissue with 50 microliters of mounting medium. Using a pipette with a cut tip, carefully transfer the ovaries onto a microscope slide.

Under the stereo microscope, and using fine nosed forceps, completely separate each ovariole. Remove the connecting tissues at the posterior and stage 14, or mature egg chambers, leaving the young, transparent egg chambers at the interior. Using the forceps, carefully align the egg chambers so that they do not overlap.

Slowly lower a number 1.5 glass cover slip onto the samples, and allow the mounting medium to polymerize for several hours at room temperature. Seal the edges with transparent nail polish and image the samples, or store them at four degrees Celsius in dark conditions until needed. The slides can be stored for about two weeks.

Visualize the slides under a confocal microscope using a 63x oil immersion objective lens, and capture three-dimensional Z-stack images of the ovarian tissue. The drosophila ovarioles shown here, displaying the typical successive developmental stages of egg chambers from anterior to posterior. Confocal images show EdU staining of the punctate structure associated with mitochondria, and EdU is confirmed to be incorporated into the MT DNA and nuclei during drosophila oogenesis.

Here MT DNA replication in the drosophila germarium is visualized by EdU incorporation in the presence of the nuclear DNA polymerase inhibitor aphidicolin. Under these conditions, nuclear incorporation of EdU is shown to be reduced, and many puncta were localized within mitochondria. In the absence of aphidicolin treatment, MT DNA replication is detected at a very low level in the drosophila germarium.

A confocal section of a wild type germarium shows intense EdU incorporation in the nuclei, with MT DNA puncta barely detectable. When wild type germaria are treated with a mitochondrial uncoupler, FCCP, at varying concentrations, MT DNA replication is impaired. This is corroborated by a reduction in EdU staining, as seen in the two micromolar, five micromolar, and 10 micromolar treatments.

Following this procedure, simple quantification of mitochondrial DNA replication under various genetic and pharmacological preparations can be done.