This work was supported by Department of Defense start-up funds to J.T.S.

1. Prepare Animals, Tools, and Media for Dissection
<ol>
	<li>Cross male and female flies to obtain progeny of the desired genotype. Useful transgenic markers for identification and staging of meiotic spermatocytes include GFP-tubulin to label the meiotic spindle and RFP-histone 2A to label chromsomes8. Use five to ten females per cross and maintain crosses on standard fly food in vials in a 25 &#176;C incubator until third instar larvae begin crawling up the sides of the vial (4&#8211;5 days). ...

<ol>
	<li>Khodjakov, A., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+division+process+in+living+tissue+culture+cells.">Imaging the division process in living tissue culture cells.</a> Methods. 38 (1), 2-16 (2006).</li><li>Ong, J. Y., Torres, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+mechanisms+of+cell+division.">Dissecting the mechanisms of cell division.</a> The Journal of Biological Chemistry. 294 (30), 11382-11390 (2019).</li><li>Levine, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycling+through+development.">Cell cycling through development.</a> Development. 131 (10), 2241-2246 (2004).</li><li>Gross, S. M., Rotwein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+Growth+Factor+Signaling+and+Cell+Cycle+Progression+in+Individual+Fibroblasts.">Unraveling Growth Factor Signaling and Cell Cycle Progression in Individual Fibroblasts.</a> The Journal of Biological Chemistry. 291 (28), 14628-14638 (2016).</li><li>Mittelman, D., Wilson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fractured+genome+of+HeLa+cells.">The fractured genome of HeLa cells.</a> Genome Biology. 14 (4), 111 (2013).</li><li>Bury, L., Coelho, P. A., Glover, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Meiosis+to+Mitosis:+The+Astonishing+Flexibility+of+Cell+Division+Mechanisms+in+Early+Mammalian+Development.">From Meiosis to Mitosis: The Astonishing Flexibility of Cell Division Mechanisms in Early Mammalian Development.</a> Current Topics in Developmental Biology. 120, 125-171 (2016).</li><li>Giansanti, M. G., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+Drosophila+spermatocytes+tell+us+about+the+mechanisms+underlying+cytokinesis.">What Drosophila spermatocytes tell us about the mechanisms underlying cytokinesis.</a> Cytoskeleton. 69 (11), 869-881 (2012).</li><li>Karabasheva, D., Smyth, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+dynein-independent+mechanism+focuses+the+endoplasmic+reticulum+around+spindle+poles+in+dividing+Drosophila+spermatocytes.">A novel, dynein-independent mechanism focuses the endoplasmic reticulum around spindle poles in dividing Drosophila spermatocytes.</a> Scientific Reports. 9 (1), 12456 (2019).</li><li>Polevoy, G., 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=Dual+roles+for+the+Drosophila+PI+4-kinase+four+wheel+drive+in+localizing+Rab11+during+cytokinesis.">Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis.</a> Journal of Cell Biology. 187 (6), 847-858 (2009).</li><li>Rebollo, E., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+noncentrosomal+microtubules+to+spindle+assembly+in+Drosophila+spermatocytes.">Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.</a> PLoS Biology. 2 (1), 8 (2004).</li><li>Smyth, J. T., Schoborg, T. A., Bergman, Z. J., Riggs, B., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proper+symmetric+and+asymmetric+endoplasmic+reticulum+partitioning+requires+astral+microtubules.">Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules.</a> Open Biology. 5 (8), (2015).</li><li>Demarco, R. S., Eikenes, &. #. 1. 9. 7. ;. H., Haglund, K., Jones, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+spermatogenesis+in+Drosophila+melanogaster.">Investigating spermatogenesis in Drosophila melanogaster.</a> Methods. 68 (1), 218-227 (2014).</li><li>Fuller, M. T., Bate, M., Martinez-Arias, A. <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. , 71-147 (1993).</li><li>Dunleavy, E. M., 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=The+Cell+Cycle+Timing+of+Centromeric+Chromatin+Assembly+in+Drosophila+Meiosis+Is+Distinct+from+Mitosis+Yet+Requires+CAL1+and+CENP-C.">The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C.</a> PLOS Biology. 10 (12), 1001460 (2012).</li><li>Fabian, L., Brill, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+spermiogenesis:+Big+things+come+from+little+packages.">Drosophila spermiogenesis: Big things come from little packages.</a> Spermatogenesis. 2 (3), 197-212 (2012).</li><li>Ramm, S. A., Scharer, L., Ehmcke, J., Wistuba, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+competition+and+the+evolution+of+spermatogenesis.">Sperm competition and the evolution of spermatogenesis.</a> Molecular Human Reproduction. 20 (12), 1169-1179 (2014).</li><li>Savoian, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopy+Methods+for+Analysis+of+Spindle+Dynamics+in+Meiotic+Drosophila+Spermatocytes.">Microscopy Methods for Analysis of Spindle Dynamics in Meiotic Drosophila Spermatocytes.</a> Methods in Molecular Biology. 1471, 265-276 (2017).</li><li>Sitaram, P., Hainline, S. G., Lee, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+analysis+of+spermatogenesis:+live+and+fixed+preparations+of+Drosophila+testes.">Cytological analysis of spermatogenesis: live and fixed preparations of Drosophila testes.</a> Journal of Visualized Experiments. (83), e51058 (2014).</li><li>Zamore, P. D., Ma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Drosophila+melanogaster+testes.">Isolation of Drosophila melanogaster testes.</a> Journal of Visualized Experiments. (51), (2011).</li><li>Lerit, D. A., Plevock, K. M., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+larval+neuroblasts.">Live imaging of Drosophila larval neuroblasts.</a> Journal of Visualized Experiments. (89), (2014).</li><li>Belloni, G., 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=Mutations+in+Cog7+affect+Golgi+structure,+meiotic+cytokinesis+and+sperm+development+during+Drosophila+spermatogenesis.">Mutations in Cog7 affect Golgi structure, meiotic cytokinesis and sperm development during Drosophila spermatogenesis.</a> Journal of Cell Science. 125 (22), 5441-5452 (2012).</li><li>Harel, A., 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=Persistence+of+major+nuclear+envelope+antigens+in+an+envelope-like+structure+during+mitosis+in+Drosophila+melanogaster+embryos.">Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos.</a> Journal of Cell Science. 94 (3), 463-470 (1989).</li><li>Katsani, K. R., Karess, R. E., Dostatni, N., Doye, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+Drosophila+nuclear+envelope+components.">In vivo dynamics of Drosophila nuclear envelope components.</a> Mol Biol Cell. 19 (9), 3652-3666 (2008).</li><li>Tates, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytodifferentiation during spermatogenesis in Drosophila melanogaster: An electron microscope study. , (1971).</li><li>Gartner, S. M., Rathke, C., Renkawitz-Pohl, R., Awe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+Drosophila+pupal+testis+and+single+male+germ-line+cysts:+dissection,+imaging,+and+pharmacological+treatment.">Ex vivo culture of Drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment.</a> Journal of Visualized Experiments. (91), 51868 (2014).</li><li>Ohkura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis:+an+overview+of+key+differences+from+mitosis.">Meiosis: an overview of key differences from mitosis.</a> Cold Spring Harbor perspectives in biology. 7 (5), (2015).</li><li>Friedman, J. R., Webster, B. M., Mastronarde, D. N., Verhey, K. J., Voeltz, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+sliding+dynamics+and+ER-mitochondrial+contacts+occur+on+acetylated+microtubules.">ER sliding dynamics and ER-mitochondrial contacts occur on acetylated microtubules.</a> Journal of Cell Biology. 190 (3), 363-375 (2010).</li><li>Bonaccorsi, S., Giansanti, M. G., Cenci, G., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methanol-acetone+fixation+of+Drosophila+testes.">Methanol-acetone fixation of Drosophila testes.</a> Cold Spring Harbor Protocols. 2011 (10), 1270-1272 (2011).</li><li>Bonaccorsi, S., Giansanti, M. G., Cenci, G., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraformaldehyde+fixation+of+Drosophila+testes.">Paraformaldehyde fixation of Drosophila testes.</a> Cold Spring Harbor Protocols. 2012 (1), 102-104 (2012).</li></ol>

The authors have nothing to disclose.

We have described a protocol for the preparation of larval or early pupal Drosophila testes, optimized for long-term, live imaging of spermatocyte cell division. This is a powerful method for analysis of cell division in the physiological context of intact tissue. The power of this method is further expanded when combined with Drosophila genetic tools, such as specific gene mutations, tissue-specific RNAi-mediated suppression, and fluorescently labeled protein and organelle markers. In addition, the sma...

Cell division is often studied using cell lines grown in culture1. While we have gained a wealth of invaluable insight and understanding of fundamental mechanisms from these studies2, cells grown in culture cannot fully recapitulate the physiology of cell division as it occurs in intact, living tissue. For example, in intact tissues and organs, cells must divide at the right place and at the right time so that progeny cells are properly situated within the tissue, so that they can undergo appropriate differentiation or functional programs, and so that cell proliferation is properly coordinated with tissue growth or homeostasis3. For cells grown in culture on the other hand, cell division is generally regulated by growth factors in the culture medium4, and thus we cannot learn from these cells how in vivo environmental factors such as tissue architecture or developmental signaling influence the division process. It is also important to note that many of the cell lines used to study cell division, such as HeLa and U2OS cells, were derived from metastatic tumors5. Therefore, many aspects of the basic physiology of these cancer cells, such as cell cycle regulatory mechanisms and chromosomal stability, have likely been altered compared to healthy cells. Complete understanding of cell division physiology, therefore, depends on our ability to study dividing cells in their native, in vivo environments that preserve physiological regulatory mechanisms and tissue architecture.
Advances in understanding how cell division operates within intact tissues and organs are hampered by difficulties inherent to in vivo or ex vivo analysis. First, it can be difficult or impossible to access dividing cells for microscopic analysis within large organs or thick tissues. Second, it is often difficult to predict when individual cells will divide in vivo. Third, tissue physiology may rapidly deteriorate during ex vivo culture. In this protocol, we describe readily accessible methods for live and fixed analysis of Drosophila melanogaster spermatocytes as they undergo meiotic cell divisions within fully intact testes. These cells are ideal for live, ex vivo analysis because they are readily accessible with standard optical methods such as confocal microscopy, they divide at predictable times and locations, and intact testes can be maintained in ex vivo culture for up to about 24 h. In addition, Drosophila spermatocytes are large round cells (approximately 20&#8211;30 &#181;m in diameter) that do not change shape when they divide, making them ideal for high resolution, time-lapse imaging of cellular components such as the spindle apparatus and cytoplasmic organelles. Although these cells undergo meiosis as opposed to mitosis, many of the essential cell division processes are very similar between these two cell division mechanisms6. These advantages, combined with powerful Drosophila genetic tools, have made Drosophila spermatocytes an extensively used model for ex vivo analysis of essential cell division processes including spindle formation and regulation, cytokinesis, and organelle remodeling and partitioning7,8,9,10,11.
Spermatocytes are cells that are at the meiotic stage of spermatogenesis. In Drosophila, spermatogenesis begins with a group of germline stem cells that are located in a small region or &#8220;hub&#8221; at one pole of the testis12,13. These cells divide by an asymmetric mitosis, giving rise to a single differentiated spermatogonium. Spermatogonia then undergo four synchronous mitoses to produce a group of 16 cells that remain closely associated within a single cyst. At this stage, the cells transition from a mitotic to a meiotic cell cycle and are referred to as spermatocytes. Spermatocytes spend about two to three days in an extended G2 stage of the cell cycle, during which they grow dramatically and undergo cytological changes in preparation for the two meiotic divisions and subsequent spermiogenesis13,14. The entire group of 16 spermatocytes within a single cyst then enter the first meiotic division at the same time. Thus, several meiotic spermatocytes can be imaged simultaneously as they proceed through cell division. The first meiotic division proceeds for approximately 1.5 h and is followed almost immediately by the second meiotic division, yielding 64 total spermatids that go on to differentiate into mature spermatozoa.
A unique advantage of using spermatocytes to study cell division in live, intact tissue is that groups or cysts of cells constantly progress through the different stages of spermatogenesis, and cells at all stages of spermatogenesis can usually be identified in any given testis (see Figure 3A). Therefore, it is relatively easy to find meiotic cells in whole testes. We generally focus our analyses on the first as opposed to second meiosis because these cells are much larger and more amenable to high resolution imaging, but the entire process encompassing both meiotic divisions can be successfully imaged. It should also be noted that the general protocol for testis preparation and culture can be used to analyze other processes of spermatogenesis as well, such as the earlier mitotic stem cell or spermatogonial divisions or the cytological changes that occur as spermatids mature into spermatozoa15. Many of these aspects of spermatogenesis are highly conserved between Drosophila and humans16.
Drosophila spermatocytes begin to reach the meiotic phase of spermatogenesis during the third instar larval stage of Drosophila development13. Therefore, testes isolated from lifecycle stages beginning with third instar larvae and including pupae and adults can be used for analysis of dividing spermatocytes. Several excellent protocols are available for extraction of testes from adult male flies for live and fixed analysis of spermatogenesis17,18,19. These protocols may be preferable for studying late stages of spermatogenesis or if genetic markers that are only visible in adults must be used. The protocol focuses instead on testis preparation from larvae and early pupae, because testes at these stages have several advantages that are specifically pertinent to cell division analysis by high resolution, time-lapse microscopy. First, testes from larvae and pupae are smaller than those from adults, and the cells within the organ are less crowded. Because of this, dividing spermatocytes can often be imaged near the outer surface of larval testes, without having to penetrate through multiple layers of light scattering tissue. Second, adult testes move rhythmically due to contractions of the attached accessory organs, and these movements make time-lapse imaging of individual cells challenging. And third, larval testes are advantageous when studying gene mutations that cause pupal or adult lethality. Our methods are optimized for long-term culture of testes on the microscope stage, allowing for imaging of multiple rounds of cell division or the progression of individual cells through multiple stages of spermatogenesis in the same preparation. We also describe a protocol for fixation and immunostaining of whole testes. Overall, our protocols are particularly useful for those interested in studying cell division in intact tissue, and the ability to combine spermatocyte analysis with highly tractable Drosophila genetic tools makes this an especially powerful approach.

<table>
	<tbody>
		<tr>
			<td>5&#34; Dissecting Probe</td>
			<td>Fisher</td>
			<td>08-965-A</td>
			<td></td>
		</tr>
		<tr>
			<td>5.75&#34; Glass Pasteur Pipet</td>
			<td>Fisher</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher</td>
			<td>BP9703-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Straight forcepts with fine tips</td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>Fisher</td>
			<td>12-544-2</td>
			<td>Slides for mounting fixed tissue, with frosted writing surface</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma</td>
			<td>H8898-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumox Dish 50</td>
			<td>Sarstedt</td>
			<td>SAR946077410</td>
			<td>Gas-permeable tissue culture dish</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher</td>
			<td>12-541-B</td>
			<td>22x22 mm, #1.5 glass coverslip</td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-15</td>
			<td>Insect pins used to make scalpel tool</td>
		</tr>
		<tr>
			<td>Nickel Plated Pin Holder</td>
			<td>Fine Science Tools</td>
			<td>26018-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 32% Solution, EM Grade</td>
			<td>Electron Microsocopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Beveled Edge Microscope Slides</td>
			<td>Fisher</td>
			<td>12-549-5</td>
			<td>Slides used for dissections</td>
		</tr>
		<tr>
			<td>PYREX Spot Plates</td>
			<td>Fisher</td>
			<td>13-748B</td>
			<td>9-well glass dissecting dish</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila medium</td>
			<td>Fisher</td>
			<td>21720-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.22 &#181;m</td>
			<td>EMD Millipore</td>
			<td>SLGS033SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Microscopy mounting medium</td>
		</tr>
	</tbody>
</table>

preparation of drosophila larval and pupal testes for analysis of cell division in live, intact tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

When this protocol is successfully executed, testes will remain fully intact for imaging by confocal microscopy or other fluorescence microscopy methods. As seen in Figure 3A, the cellular organization of the testes is preserved, and the progression of cell differentiation from one end of the testis to the other including spermatogonia, spermatocytes, and haploid spermatids is visible. GFP-tubulin is a useful marker for identifying dividing spermatocytes and for live imaging of the progressi...

Watch this Scientific Journal Video about Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue at JoVE.com

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

This protocol can be used to analyze the mechanisms of cell division in the context of living, intact tissue using fluorescence microscopy. The main advantage of this technique is that cells'native physiological environments are preserved, while also allowing for live imaging over long time courses. The most important part of this technique is making sure that the testes are not damaged during dissection or mounting.This is a skill that is acquired with a little bit of time and practice. Visual demonstration of this method is critical, because it shows how to identify and isolate the testes, and how to mount them for imaging without damaging the tissue. Prepare animals, tools, and media as described in the manuscript.Before beginning the dissection, use the media filled filter syringe to expel three drops of media each 50 microliters at the top, middle, and bottom of a glass slide. Using a dissecting probe, transfer five to 10 third instar larvae or early pupae to the top drop of media on the slide. Gently agitate the larvae and pupae in the media with the dissecting probe to remove any food or debris.Use the dissecting probe to turn a single larvae or pupae on its side. Look for the two bilateral testes which appear as translucent oval structures on the posterior third of the body. Animals that lack testes are female, and should be discarded.When a male larvae or pupae is found and is clean, move it to the second drop of media. Repeat until three or four male larvae or pupae have been transferred to the second drop of media. Then working in the second drop of media, grasp a single animal with a pair of forceps at its mid-region, just anterior to the testes.Use a second pair of forceps to gently tear the animal in half. Using a pair of forceps, hold the posterior end of the animal down on the glass slide. Starting just next to the forceps, push down on the cuticle using the edge of the scalpel.Move the scalpel toward the cut end of the animal. This will expel the animal's internal organs, which include the guts, fat bodies, and testes. The testes are colorless and nearly transparent oval organs embedded in ribbons of fat body.Gently tease apart the testes from the rest of the organs. To transfer the testes one at a time, insert the edge of the scalpel under the fat body, lift the tissue out of the media, and quickly move it to the third drop. If there is not enough fat body still attached to the testes to lift the tissue with the scalpel use a glass Pasteur pipette that has been prewetted with dissection medium.Working in the third drop of medium, use the sharp end of the scalpel to gently slice away excess fat body from the testes, leaving just a small rim of fat body around the edges. First, use the filter syringe to deposit a single drop of Schneider's medium about 30 microliters onto the center of a 50 millimeter gas permeable culture dish. Using the scalpel tool or a prewetted Pasteur pipette transfer up to five of the prepared testes to the drop on the dish.Next, use the scalpel tool to gently push the testes down onto the gas permeable membrane, and into the center of the drop of medium. Using a one milliliter syringe, place four drops of halocarbon oil on the gas permeable membrane surrounding the drop of medium. The locations of the drops should correspond to the four corners of a 22 millimeter class coverslip.Then align the corners of a 22 millimeter glass coverslip with the four drops of halocarbon oil and gently lower the coverslip onto the media and oil. Allow the coverslip to settle, and for the media containing the testes the spread between the drops of oil. Place the culture dish under a dissecting microscope.To remove excess media, insert the corner of a delicate task wipe under the coverslip. Wick away enough media for the glass coverslip to just make contact with the surface of the largest testis. Do not remove too much media and lower the coverslip too far.This will exert pressure on the testes and may cause them to rupture. Finally, place a drop of immersion oil on the glass coverslip just above the testes. Turn over the dish, and place it on the microscope stage.Move the 40 or 60 X microscope objective into the oil until it is just below the testes. Use transmitted light to find a single testis and bring it into focus. Place 0.5 milliliters of 8%paraformaldehyde in PBSTx in one well of a nine well dissecting dish.Use a glass Pasteur pipette to transfer up to 10 of the testes to the well. Ensure that the testes are lying on the bottom of the well. After 20 minutes, transfer the testes to a well containing 0.5 milliliters of PBSTx.Wash the testes by agitating gently for five minutes. Wash two more times in new wells with fresh PBSTx. Prepare a dilution of the primary antibody in a 1.5 milliliter microcentrifuge tube as described in the manuscript.Then transfer the testes from the nine well glass dissecting dish to the tube. Incubate the tube overnight at four degrees Celsius with gentle rocking or nutation. The procedure for staining the testes with the secondary antibody is similar.See the manuscript for details. First, using a Pasteur pipette, transfer the testes from the nine well plate onto a glass microscopy slide. If necessary, use the edge of the scalpel to push the testes down onto the surface of the slide.Next, use the corner of a delicate task wipe to wick away excess liquid from the testes. Remove as much liquid as possible. Then apply a 30 to 50 microliter drop of microscopy mounting medium to the testes.Finally, place a 22 millimeter coverslip onto the drop of mounting medium. Allow the coverslip to settle, and the mounting medium to spread under the coverslip. If necessary, apply gentle pressure to the coverslip to squeeze out excess mounting medium and allow the coverslip to rest on the surface of the testes.When this protocol is successfully executed, the cellular organization of the testes is preserved, and the progression of cell differentiation from one end of the testis to the other is visible. Spermatocytes about to begin meiosis and those in metaphase can be identified. In testes prepared using this protocol, live imaging analysis shows the dynamic reorganization of the endoplasmic reticulum and micro tubules in dividing spermatocytes.As described in the protocol, great care must be taken not to apply pressure that causes the testes to burst. Cysts of spermatocytes rapidly flow out of a ruptured testis, making live time lapse imaging difficult. This protocol is optimized for imaging dividing spermatocytes in living, intact tissue.Therefore, it is essential that the testes are not damaged during dissection or mounting. Our method should also be suitable for super resolution imaging techniques, such as structured illumination microscopy, providing new insights into dynamic sub-cellular processes that govern cell division.

preparation-drosophila-larval-pupal-testes-for-analysis-cell-division

Research

JoVE Journal

Developmental Biology

7.9K vues.  Uniformed Services University of the Health Sciences. Ce protocole peut être utilisé pour analyser les mécanismes de la division cellulaire dans le contexte des tissus vivants intacts à l’aide de microscopie de fluorescence. Le principal avantage de cette technique est que les environnements physiologiques indigènes des cellules sont préservés, tout en permettant l’imagerie en direct sur de longs cours de temps. La partie la plus importante de cette technique est de s’assurer que les testicules ne sont pas endommagés pendant la dis...