We thank K. Delaney for comments on the manuscript. This work was supported by the National, Heart, Lung, and Blood Institute (NHLBI) Intramural Program.

1. Tissue Collection and Dissection
<ol>
	<li>In each vial containing dry yeast, culture 10 adult female flies with 10 males for 2-3 days. 
	Note: Maintaining well-fed female flies will improve the overall quality and yield of ovary production and facilitate dissection.</li>
	<li>Anesthetize the female flies on a carbon dioxide (CO2) fly pad.</li>
	<li>Under the stereoscope, place several drops of RT medium (Schneider&#39;s Drosophila medium supplemented with 10% fetal bovine serum (FBS)) on a dissecting pad. Conduct all the dissection procedures in the medium to keep the tissues alive and healthy.</li>
	<li>Grab a fattened female fly with sharp fine-nosed forceps at its lower thorax. Use another set of forceps to tug gently at the extreme posterior of the fly until the tissues in the abdomen are exposed. Detach the two ovaries from other tissues (e.g. guts). 
	Note: Each ovary displays an opaque structure that is composed of 16-20 attached ovarioles.</li>
	<li>To enhance the penetration of reagents, open the ovaries by pulling them apart and passing the tips of the forceps in between each ovariole a couple of times. Keep the ovarioles connected within an ovary to minimize loss of tissues during the following procedures.</li>
	<li>Transfer the ovaries immediately to an 1.5 ml microcentrifuge tube containing 500 &#181;l of Schneider&#39;s Drosophila medium with 10% FBS.</li>
	<li>Repeat the dissection and collect 10-15 ovaries in each microcentrifuge tube.</li>
</ol>
2. EdU Labeling
<ol>
	<li>Aspirate the medium in each tube, and replace with 500 &#181;l of Schneider&#39;s Drosophila medium with 10% FBS containing 7 &#181;M aphidicolin. 
	Note: Aphidicolin is used to block nuclear DNA synthesis by inhibiting DNA polymerase &#945; without affecting mtDNA replication 7,12. It is possible to increase the aphidicolin concentration to up to 70 &#181;M to achieve better inhibition. The stock solution of 3-30 mM aphidicolin in DMSO can be stored in the dark at -20 &#176;C for up to 6 weeks.</li>
	<li>To keep the ovaries healthy, ensure that they are immersed under solutions while changing the medium. Use Schneider&#39;s Drosophila medium with 10% FBS through step 2.6.</li>
	<li>Incubate ovaries for 3 hr at RT on a bench-top rocker with gentle rotation.
	<ol>
		<li>For drug treatment, e.g.,&#160;mitochondrial uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), add the appropriate concentration of drug (e.g.,&#160;10 &#181;M FCCP) into the medium after 2 hr of aphidicolin treatment. Continue incubating for another 1 hr.</li>
	</ol>
	</li>
	<li>Remove the medium containing aphidicolin with or without drug. Briefly rinse the ovaries with medium (aphidicolin is not necessary) twice.</li>
	<li>Add 1 ml medium containing 10 &#181;M EdU and 7 &#181;M aphidicolin and continue incubating at RT for 2 hr. Store the 10 mM EdU stock solution (in DMSO) at -20 &#176;C.</li>
	<li>Remove the medium containing EdU and aphidicolin. Wash with medium (without aphidicolin) twice for 3 min each.</li>
</ol>
3. Tissue Fixation and Permeabilization
<ol>
	<li>Prepare a 4% paraformaldehyde solution in phosphate buffered saline (PBS, pH 7.4). 
	Note: We used commercial available paraformaldehyde stored in pre-scored ampoules. Open a new ampoule and dilute to 4% before use. 
	CAUTION: Formaldehyde is toxic; it should be handled in a fume hood with skin and eye protection.</li>
	<li>Fix ovaries with 4% paraformaldehyde for 20 min at RT with gentle rotation.</li>
	<li>Remove the fixative and wash ovaries twice in 1 ml of 3% BSA in PBS for 5 min each time.</li>
	<li>To permeabilize the tissues, remove the wash solution and add 1 ml of 0.5% Triton X-100 in PBS. Incubate at RT for 20 min.</li>
	<li>Remove the permeabilization solution and wash twice in 1 ml of 3% BSA in PBS.</li>
</ol>
4. EdU Detection
Note: EdU detection is based on &#34;click&#34; chemistry, a Cu(I)-catalyzed [3+2] cycloaddition 13, which adds fluorescent azides to the terminal alkyne group of EdU, and the fluorescent molecule is subjected to subsequent detection. Since Cu(I), which is easily oxidized to the non-catalytic Cu(II) species, is required to catalyze the reaction, it is recommended to reduce the Cu(II) sulfate in situ to obtain Cu(I). That is, Cu(II) sulfate is used in the presence of a reductant such as ascorbic acid (herein the &#34;buffer additive&#34;) to generate copper (I).
<ol>
	<li>Before the experiment, prepare the working solution of the Alexa Fluor azide (Alexa Fluor 488 azide, Alexa Fluor 555 azide or others, depending on the preferred fluorophore, which are named as &#34;dye azide&#34; hereafter), EdU reaction buffer and EdU buffer additive according to manufacturer&#39;s instructions.
	<ol>
		<li>Reconstitute the dye azide in 70 &#181;l DMSO. Store the working solution at -20 &#176;C for up to 1 year.</li>
		<li>Prepare fresh EdU reaction buffer by diluting the 10x EdU reaction buffer with deionized water. 
		Note: After use, store any remaining 1x solution at 4 &#176;C. The 1x solution is stable for up to 6 months.</li>
		<li>Make a 10x stock solution of the EdU buffer additive by fully dissolving the powder in 2 ml deionized water. 
		Note: This stock solution is stable for up to 1 year at -20 &#176;C. If the solution develops a brown color, it has degraded and should be discarded.</li>
	</ol>
	</li>
	<li>Prepare the EdU reaction cocktail fresh each time, and use within 15 min of preparation. To achieve reproducible results, make sure the reaction components are maintained at the same ratios.
	<ol>
		<li>Make fresh 1x EdU buffer additive solution by diluting the 10x stock solution (prepared in step 4.1.3) 1:10 in deionized water. Use this solution on the same day.</li>
		<li>Prepare 1 ml of EdU reaction cocktail by combining the following ingredients in order: 860 &#181;l 1x EdU reaction buffer, 40 &#181;l CuSO4, 2.5 &#181;l dye azide, 100 &#181;l 1x EdU buffer additive. 
		Note: It is important that the ingredients are added in order to ensure optimal performance.</li>
	</ol>
	</li>
	<li>Remove the PBS with 3% BSA and add 0.5 ml of the EdU reaction cocktail to each tube. Incubate at RT for 30 min with gentle rotation. Keep the samples protected from light.</li>
	<li>Remove the EdU reaction cocktail and wash once with 1 ml of 3% BSA in PBS.</li>
	<li>Wash the samples once with 1 ml of PBS.</li>
</ol>
5. Antibody Labeling
Note: Perform antibody labeling after EdU staining. If no additional staining is desired, one can proceed to mounting and imaging directly. It is important that the samples be protected from light in all the following procedures.
<ol>
	<li>Remove the wash solution. Block the ovaries in 1 ml of blocking solution containing 0.2% BSA and 0.1% Triton X-100 in PBS for 30 min.</li>
	<li>Remove the blocking solution and replace with primary antibody diluted in blocking solution (e.g., mouse ATP synthase subunit &#945; antibody, 1:1,000 dilution). Incubate in the dark at 4 &#176;C O/N.</li>
	<li>Wash the samples with 1 ml of blocking solution 3 times, 10 min each time. To minimize the background, wash two more times for 30 min each. Remove the blocking solution.</li>
	<li>Incubate with secondary antibody diluted in blocking solution (e.g., goat anti-mouse Alexa Fluor 568 secondary antibody, 1:200 dilution) for 2 hr at RT. 
	Note: Use a different color than the one coupled to EdU.</li>
	<li>Repeat the wash steps performed in step 5.3.</li>
	<li>Rinse with 1 ml of PBS once to remove the detergent.</li>
</ol>
6. Mounting and Imaging
<ol>
	<li>Carefully remove all the PBS and immediately cover ovaries with 50 &#181;l mounting medium (with or without DAPI). 
	Note: Use a mounting medium that does not harden while the ovarioles are being separated from each other. Ovaries can be stored in mounting medium at 4 &#176;C for up to a week.</li>
	<li>Cut the end of a pipette tip and use it to transfer ovaries carefully onto a microscope slide.</li>
	<li>Completely separate each ovariole with sharp fine-nosed forceps under the stereomicroscope. Remove the connecting tissues at the posterior end and stage 14 or mature egg chambers. The young egg chambers are at the anterior end and transparent.</li>
	<li>Align the egg chambers with forceps so that they do not overlap with each other.</li>
	<li>Slowly lower a # 1.5 glass coverslip on top of the samples. Allow the mounting medium to polymerize for several hours at RT. Seal with transparent nail polish.</li>
	<li>Image slides the next day, or store at 4 &#176;C prior to imaging.</li>
	<li>Visualize under a confocal microscope with a 63X oil-immersion objective. Capture three-dimensional z-stacks images. 
	Note: There is strong EdU signal in later-stage egg chambers and background fluorescence in the epithelial sheath. When examining EdU labeling in a particular germarium, one should avoid germariums that are overlapped by or close to other tissues or later-stage egg chambers.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of Drosophila melanogaster. , (1993).</li><li>Riechmann, V., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axis+formation+during+Drosophila+oogenesis.">Axis formation during Drosophila oogenesis.</a> Curr Opin Genet Dev. 11 (4), 374-383 (2001).</li><li>Lin, H., Yue, L., Spradling, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+views+of+the+biochemistry+of+eucaryotic+DNA+replication+revealed+by+aphidicolin,+an+unusual+inhibitor+of+DNA+polymerase+alpha.">New views of the biochemistry of eucaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase alpha.</a> Cell. 23 (3), 647-648 (1981).</li><li>McClung, C., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+behavioral+responses+to+free-base+cocaine+and+the+development+of+behavioral+sensitization+in+Drosophila.">Stereotypic behavioral responses to free-base cocaine and the development of behavioral sensitization in Drosophila.</a> Curr Biol. 8 (2), 109-112 (1998).</li><li>Moore, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93 (6), 997-1007 (1998).</li><li>Dzitoyeva, S., Dimitrijevic, N., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-aminobutyric+acid+B+receptor+1+mediates+behavior-impairing+actions+of+alcohol+in+Drosophila:+adult+RNA+interference+and+pharmacological+evidence.">Gamma-aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in Drosophila: adult RNA interference and pharmacological evidence.</a> Proc Natl Acad Sci U S A. 100 (9), 5485-5490 (2003).</li><li>Koch, E. 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The authors have nothing to disclose.

EdU labeling is a novel and efficient method to detect DNA synthesis in proliferating cells, which is based on the incorporation and staining of nucleoside analogues in newly synthesized DNA. This method is superior to the BrdU labeling method in that it is faster and highly sensitive. More importantly, it allows for good structural preservation and efficient EdU-dye penetration of whole-mount tissues 9, 10. Historically, as a superior alternative to BrdU labeling, EdU labeling was used for studying nuclear DNA replication during the S-phase of the cell cycle. Aphidicolin is an inhibitor of DNA polymerase &#945;, which is the main polymerase for nuclear DNA replication at S-phase 1215. mtDNA replication is carried out by DNA polymerase &#947;, which is insensitive to aphidicolin treatment. Hence, treatment with aphidicolin prior to and during the EdU incubation significantly inhibited EdU incorporation into nuclear DNA. It should be noted that aphidicolin may be stable for several weeks if appropriately stored, and the efficacy of aphidicolin in inhibiting nuclear DNA replication was variable in our hands. The ovarioles or egg chambers with strong nuclear DNA labeling should be excluded from further data analyses.
mtDNA replication can be readily visualized as puncta in the cytoplasm, which also affords a straightforward way to quantify the level of mtDNA replication by counting the number of EdU puncta, normalized to the total volume of cytoplasm. Imaging software can be applied to automatically identify EdU puncta in microscopic images, which is especially useful for computational analyses of large data sets. However, precautions should be taken, because the incomplete inhibition of nuclear DNA replication can lead to the EdU incorporation in distinct loci on the chromosome and display as puncta inside the nucleus. Also in other cases, the intensity of EdU incorporation into replicating mtDNA might be weak, while the background and noise could be high. Therefore, the individual parameters for automatic image analyses should be carefully defined. It is also recommended that images should be examined with trained eyes to make sure that proper EdU puncta are identified.
Visualization of newly synthesized mtDNA during Drosophila oogenesis provides an opportunity to investigate how mtDNA replication is regulated under physiological or pathological conditions, by carrying out the experiment in flies subjected to a variety of pharmacological or genetic perturbations. In a previous study, EdU incorporation assay was performed in a mtDNA mutant Drosophila mt:CoIT300I11. Furthermore, to disrupt the mitochondrial membrane potential, ovaries were treated with various concentrations of mitochondrial uncoupler FCCP or 2,4-dinitrophenol (DNP) prior to EdU incorporation. Depending on the experimental purposes and drug characteristics, different methods might be adopted for effective delivery. For adult flies, drugs can be presented as a vapor (e.g., ethanol and cocaine) 16,17 or drugs can be injected into the abdomen, where it quickly diffuses throughout the body 18. The most common practice is that drugs are added to the fly food or a sucrose/drug-saturated filter paper. For example, the inhibitor of microtubule assembly, colchicine, was fed to flies for 2-3 days before ovary dissection 19. Hence, it is important to evaluate the drug delivery method and choose appropriate concentrations.
To ensure the successful imaging of mtDNA replication in Drosophila ovaries, several critical steps must be carefully executed. Foremost, preserving the viability and health of the ovaries during dissection and EdU incorporation is essential (steps 1 and 2). The Schneider&#39;s Drosophila medium with FBS needs to be warmed to RT before use. One should minimize direct contact between the egg chambers and dissecting tools or pipette tips. The ovaries should be immersed under solutions all times to avoid dehydration. Mishandling the tissues can readily lead to faint or no fluorescent signals. During the tissue mounting step, ovarioles should be separated from each other and spread out on the microscope slide. Make sure the egg chambers are not stacked on top of each other. We noticed that the egg chambers beyond stage 14 displayed little EdU incorporation. Additionally, because of the large size, the late stage egg chambers often cause the neighboring younger egg chambers to be out of focus. Thus it is recommended that the late stage egg chambers be discarded.
Here we provide a detailed protocol for labeling replicating mtDNA in Drosophila adult ovaries. This method allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations, and will be useful for dissecting mechanisms underlying developmental mitochondrial biogenesis and mtDNA inheritance.

Besides the nuclear genome, each eukaryotic cell also contains thousands of copies of small circular DNA in the mitochondrial matrix. While mitochondrial DNA (mtDNA) encodes essential subunits of electron transport chain, the majority of the mitochondrial proteome, including all factors for replication and transcription of mtDNA are encoded by the nuclear genome. In contrast to the nuclear genome that follows Mendelian laws of inheritance, the animal mitochondrial genome is inherited exclusively through the maternal lineage. Therefore, understanding how mtDNA is proliferated and transmitted from one generation to the next through the female oocyte is fundamentally important. However, there is continuing debate over how mtDNA replication is regulated in the female germline. Additionally, the widely accepted mtDNA bottleneck theory for mtDNA transmission suggests that the population of mtDNA in primordial germ cells is subsampled to a relatively small number during development 12. It also implies that mtDNA replication is temporally and spatially regulated during oogenesis. Hence, the in situ detection of mtDNA replication during germline development will facilitate the understanding of the mechanism of mtDNA inheritance.
Drosophila oogenesis provides a genetically tractable system to study mtDNA replication and transmission. In each of the two Drosophila ovaries, there are 16-20 independent strings of egg chambers called ovarioles 3, which are the functional units of egg production (see Figure 1A). Each ovariole contains a progressive linear organization of oogenesis, where the anterior tip is composed of a structure called germarium. The germarium is further divided into four regions that contain germ cells at distinct developmental stages. In region 1, germline stem cells go through asymmetric division to produce daughter cells known as cystoblasts. Region 2a contains the cystoblasts that have completed their final division. Cystoblasts undergo four rounds of division producing 16-cell groups. The 16 cells remain connected to each other by cytoplasmic bridges called ring canals. Only one of the cells commits to differentiation as the oocyte, while the other 15 develop as polyploid nurse cells. An essential cytoplasmic structure, known as fusome, is proposed to facilitate with the formation of ring canals, determine the cyst polarity and the nurse cell-oocyte interactions 4,5. As the cysts move towards region 2b, the cyst structures span the entire width of the germarium, and become more associated with follicle cells. The germarium structures end with region 3 containing the first budding egg chamber. Subsequently, the egg chambers are assembled at the posterior end of the germarium, which progress through the ovariole in 14 morphologically distinct stages. The growth of oocyte depends on the nurse cells, which transport proteins, mRNA and endomembrane structures (e.g., Golgi) via the ring canals into the oocyte. During Drosophila oogenesis, it was found that a fraction of the mitochondria within each 16-cell cyst were associated with the fusome, moved through the ring canals and were delivered into the single oocyte in a large mass called Balbiani body 6. This phenomenon was proposed to contribute to the quality control of mitochondrial inheritance through female ovaries.
Detection of mtDNA synthesis relies on the incorporation of labeled DNA precursors into cellular DNA. Traditionally, a nucleoside analog of thymidine 5-bromo-2&#39;-deoxyuridine (BrdU) was used to label the mtDNA replication in tissue culture cells 7,8. However, the antibody-based BrdU labeling exhibits several limitations, especially for whole-mount tissue staining. One major disadvantage of BrdU labeling is that it requires DNA denaturation to expose the BrdU epitope, so that it can be detected by the anti-BrdU antibody. The specimen has to be treated under harsh denaturing conditions such as chemicals (e.g., hydrochloric acid or mixtures of methanol and acetic acid), heat, or digestion with DNase, which could disrupt the structure of the specimen and complicate the following staining procedure 9,10.
Here, an alternative thymidine analog 5-ethynyl-2&#180;-deoxyuridine (EdU) is used to label the replicating mitochondrial DNA in Drosophila adult ovaries. This method is faster and highly sensitive. EdU is readily incorporated into cellular DNA during DNA replication. The following detection is based on a &#34;click&#34; reaction, a Cu (I) -catalyzed covalent reaction between the terminal alkyne group and a fluorescent azide 10. Because the reaction does not require the denaturation of the specimen, it allows good structural preservation. Furthermore, the size of dye azide is only 1/500 of that of an antibody molecule 10, which enables fast and efficient penetration of whole-mount tissues. We have used this method to detect mtDNA replication during Drosophila oogenesis and found a striking spatial pattern in the germarium region of Drosophila ovary 11, which lead us to propose a replication-dependent mtDNA selective inheritance mechanism. We present here a detailed protocol on EdU labeling of mtDNA replication in Drosophila ovaries. To demonstrate the application of the protocol, we also tested the mtDNA replication in a mtDNA mutant Drosophila (mt:CoIT300I) 11, as well as with diverse treatment of mitochondrial uncouplers, which dissipate the mitochondrial membrane potential and potentially disrupt mtDNA replication.

<table><tbody><tr><td>Schneider&amp;rsquo;s Drosophila medium</td><td>Invitrogen</td><td>21720-024</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10100-147</td><td></td></tr><tr><td>Pair of Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Aphidicolin</td><td>Sigma</td><td>A0781</td><td>Aliquot after dissolving in DMSO. Avoid repetitive thawing and freezing. Protect from light. May be used within 6 weeks after dissolving.&amp;nbsp;</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C2920</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Paraformaldehyde, 16% EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Formaldehyde is toxic; it should be handled in a fume hood with skin and eye protection.</td></tr><tr><td>PBS</td><td>KD Medical</td><td>RGF-3190</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Invitrogen</td><td>C10337</td><td>EdU, CuSO&lt;sub&gt;4&lt;/sub&gt;, Alexa Fluor 488 azide, EdU reaction buffer and Edu buffer additive are included</td></tr><tr><td>Mouse ATP synthase subunit &amp;alpha; antibody, (15H4C4)</td><td>MitoSciences</td><td>Ab14748</td><td>1:1,000 dilution</td></tr><tr><td>Mouse Hts antibody (clone RC)</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>hts RC</td><td>1:1,000 dilution</td></tr><tr><td>Goat anti-mouse Alexa Fluor 568 secondary antibody</td><td>Invitrogen</td><td>A-11004</td><td>1:200 dilution</td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td></td></tr><tr><td>Glass coverslips, #1.5 22 mm x 22 mm</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Microscope slide</td><td>Fisher Scientific</td><td>22-038-103</td><td></td></tr><tr><td>Nail polish</td><td>Elf</td><td></td><td>Many of the pigments used in nail polishes are fluorescent and leach into specimens. Only clear nail polish should be used.</td></tr></tbody></table>

in situ labeling of mitochondrial dna replication in drosophila adult ovaries by edu staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

The above protocol allows visualization of the punctate structures associated with mitochondria (Figure 1B-C), which indicate mitochondrial DNA replication during Drosophila oogenesis. The EdU puncta localized with mitochondria marked by staining for ATP synthase alpha subunit (Figure 2). The observed signals were absent in ovaries treated with ethidium bromide 11, an inhibitor for mtDNA replication 14, validating that these puncta indeed label replicating mtDNA.Aphidicolin was used to inhibit nuclear DNA staining without affecting mtDNA replication (Figure 2). Without aphidicolin treatment, intense EdU signals label the nuclei, and mtDNA puncta were barely detected (Figure 3). However, in the presence of aphidicolin, nuclear incorporation was dramatically reduced and many puncta associated with mitochondria were observed.
There is a high level of mtDNA replication in post-germarium egg chambers (Figure 1B). However, notably, mtDNA replication displayed a spatial pattern in the germarium. As indicated by the number of EdU puncta, there is a moderate level of mtDNA replication in region 1 of the germarium, but almost no EdU incorporation in region 2A (Figure 1C). As the cyst moves down to region 2B in the germarium, mtDNA replication resumed and the number of EdU puncta in the posterior cyst of region 2B was much higher than that in region 2A (Figure 1C). Specifically, intensive EdU incorporation was concentrated around the ring canals and the fusome structures, as stained by the hu li tai shao (Hts) protein. mtDNA kept replicating at a high level in region 3 of the germarium (Figure 1C)
To demonstrate the association of specific genes or treatment with mtDNA proliferation, Drosophila ovaries could be subjected to gene manipulation or drug treatment. We treated ovaries with different concentrations of FCCP, a classic mitochondrial protonophore, which dissipates mitochondrial membrane potential. As a control, DMSO had no effect on mtDNA replication (Figure 4A). High doses of FCCP (10 &#181;M) almost depleted mtDNA replication entirely throughout the germarium (Figure 4D). Nonetheless, lower concentration of FCCP (2 or 5 &#181;M) had a minor impact on mtDNA replication in region 1 but inhibited replication in regions 2B and 3 (Figure 4B-C), suggesting regions 2B and 3 are more sensitive to mitochondrial disruption, or they maintain relative slower replication kinetics. The above results indicated that mtDNA replication is associated with mitochondrial activity. Particularly, different regions of the germarium responded differently to mitochondrial impairment.
<img alt="Figure 1" src="/files/ftp_upload/54516/54516fig1.jpg" /> 
Figure 1. mtDNA replication during Drosophila oogenesis. (A) Diagram of a Drosophila ovariole and a magnified view of the germarium. The ovariole illustrates from left to right, anterior to posterior, successive developmental stages of egg chambers. In the germarium, the fusome (red), germline stem cells (blue), mitochondria (green), future oocyte (broken line, recognized by positioning, fusome structure and mitochondrial clusters), developing cysts (peach) and four developmental regions are shown. (B) Representative z-stack projection of a wild-type ovariole labeled by EdU and antibody against Hts-RC, a marker for ring canals and fusome. In the presence of aphidicolin, the EdU was incorporated into mtDNA (arrowheads) and nuclei (arrows). Scale bar, 10 &#181;m. (C) Magnified view of germaria outlined in the boxed region in B. The four developmental regions are indicated. Scale bar, 5 &#181;m 11 . <a href="https://www.jove.com/files/ftp_upload/54516/54516fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54516/54516fig2.jpg" /> 
Figure 2. mtDNA replication in the Drosophila germarium visualized by EdU incorporation with aphidicolin treatment. (A)-(D) Representative confocal section of a wild type germarium showing EdU incorporation (green, b), mitochondria, marked by ATP Synthase alpha subunit staining (red, C), and nuclei, labeled with DAPI staining (blue, D) with pre-incubation with DNA polymerase-&#945; inhibitor aphidicolin. (A&#39;-D&#39;) A magnified image of the boxed area in (A) showing EdU incorporation (B&#39;), mitochondria (C&#39;) and nuclei (D&#39;). In the presence of aphidicolin, nuclear incorporation (arrows) is reduced and many puncta were localized within mitochondria (arrowheads). Scale bars, 10 &#181;m. The figure has been modified from 11. <a href="https://www.jove.com/files/ftp_upload/54516/54516fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54516/54516fig3.jpg" /> 
Figure 3. mtDNA replication is barely detected in the Drosophila germarium without aphidicolin treatment. (A)-(D) Representative confocal section of a wild type germarium showing EdU incorporation (green), mitochondria, marked by ATP Synthase alpha subunit staining (red), and nuclei, labeled with DAPI staining (blue) in the absence of the DNA polymerase-&#945; inhibitor aphidicolin. (A&#39;-D&#39;) A magnified image of the boxed area in (A) showing EdU incorporation (B&#39;), mitochondria (C&#39;) and nuclei (D&#39;). Without aphidicolin, intense EdU signals label nuclei (arrows), and mtDNA puncta were barely detected. Scale bars, 10 &#181;m. The figure has been modified from 11. <a href="https://www.jove.com/files/ftp_upload/54516/54516fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54516/54516fig4.jpg" /> 
Figure 4. Mitochondrial uncoupler impairs the mtDNA replication. Representative z-stack projects showing wild-type germarium treated with DMSO (A) or the mitochondrial uncoupler FCCP at concentrations of 2 &#181;M (B), 5 &#181;M (C), 10 &#181;M (D) during EdU incorporation. Note the impaired EdU labeling in region 2B treated with 2 &#181;M FCCP (B), and in both region 2B and 3 treated with 5 &#181;M FCCP (outlined in boxes). Four developmental regions are shown. Arrows, nuclear DNA; arrowheads, mtDNA. Scale bar, 10 &#181;m. The figure has been modified from 11. <a href="https://www.jove.com/files/ftp_upload/54516/54516fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining at JoVE.com

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

in-situ-labeling-mitochondrial-dna-replication-drosophila-adult

Research

JoVE Journal

Genetics

9.1K Views.  National Institutes of Health. Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2´-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

Video: In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

Neuroscience

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Biology

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Developmental Biology

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Biochemistry

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

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