We thank P. Verrijzer, J. Johansen H., Okhura for antibodies and A. Debec, the Bloomington and Vienna stock centers for flies. Thanks to C. Jackson for suggestions and stimulating discussions.

1. Drosophila testes collection
NOTE: Young males (0-15 h post-eclosion) are necessary for examining diploid germ cells (i.e., spermatogonia and spermatocytes).
<ol>
	<li>Select young flies as much as possible to minimize interference from developing spermatids.</li>
	<li>Anesthetize flies by brief exposure to carbon dioxide and transfer to a fly pad14,15.</li>
	<li>Under a dissecting microscope (10x magnification), use forceps to re...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+distribution+of+nuclear+pores+during+differentiation+of+the+male+germ+cells.">Changes in distribution of nuclear pores during differentiation of the male germ cells.</a> Tissue Cell. 11 (1), 147-162 (1979).</li><li>Bolte, S., Cordelieres, F. 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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=.">.</a> Immunostaining of Drosophila testes. 2011 (10), 1273-1275 (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=.">.</a> Paraformaldehyde fixation of Drosophila testes. 2012 (1), 102-104 (2012).</li><li>White-Cooper, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis:+analysis+of+meiosis+and+morphogenesis.">Spermatogenesis: analysis of meiosis and morphogenesis.</a> Methods in Molecular Biology. 247, 45-75 (2004).</li><li>Kibanov, M. V., Kotov, A. A., Olenina, L. 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This protocol provides a method for successful immunolabeling of whole-mount adult Drosophila testes. As reported in other procedures young males must be used (we even shortened the age to less than 20 hours after eclosion) to minimize the presence of developing sperm, which risks obstructing the spermatocytes. The dissection should be rapid and the antibody dilution must be adjusted to optimize the signal-to-noise ratio21,22,23...

Drosophila testes are a valuable model for the study of nuclear architecture. In mitotic spermatogonial cells, the nuclear volume is largely occupied by chromatin. These cells undergo four rounds of division, producing a cyst of 16 primary spermatocytes. Over the next several days, the spermatocytes pass through 6 developmental stages (S1-S6), greatly increasing in volume, approximatively 20-fold1,2. They also change their nuclear morphology. The outline of the nucleus, revealed by the lamin Dm0, becomes irregular and shows deep invaginations1,3. This is accompanied by extensive gene expression changes and modifications in chromatin organization1,4,5,6,7. The interphase chromosomes are well-defined in stage S4-S5, forming three distinct masses corresponding to the 2 major bivalent autosomes and the X-Y pair docked with the nucleolus.
Mad1 was originally isolated as a key component of the mitotic checkpoint (reviewed in8). In interphase, it is described as located primarily at the nuclear envelope but also in the nucleoplasm9,10,11. In Drosophila testis, we reported that Mad1 is prominent in the nucleoplasm, intimately associated with the two major autosomal chromatin masses and to a lesser extent with the XY chromatin. We defined this chromatin-associated structure as MINT (Mad1-containing IntraNuclear Territory)12. Four other proteins were associated with MINTs in spermatocytes: the nucleoporin Mtor/Tpr, the SUMO peptidase Ulp1, the mitotic checkpoint protein Mad2 and a subunit of a Polycomb-like complex Raf212.
To complete the description of MINTs, we needed to use antibodies and were confronted with the technical problems generally encountered with immunostaining in testis: a poor permeability resulting in a significant non-uniformity of staining in the depth of the testis, particularly in the large spermatocytes. Squashed preparations increased antibody access but led to compressed images, restricting the 3D analyses, and preventing measurement of quantitative fluorescence, including colocalization. The problem of antibody penetration could also be addressed by permeabilizing agents such as 0.3% Na deoxycholate13, but this procedure did not in our hands yield reproducible results. Because we performed many co-labeling experiments, we looked to improve the permeabilization protocol. The combination of 1% NP40 plus heptane resulted in more reproducibility, particularly in studying the large spermatocytes.
This protocol performs fixation and immunostaining of testes in suspension, improving the sample quality as well as enhancing reproducibility, and rendering it suitable for confocal microscopy. We used the protocol to better define the spatial relationships of certain nuclear envelope proteins with the nucleoplasmic structures of large spermatocytes. This method will permit better investigations of the changes that accompany spermatocyte development, for example the dynamic structural changes of the nucleus including chromatin, nucleoplasm and the nuclear pores.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Drosophila srains</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ketelGFP y; ketelGFP/y+CyO</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>Gift from A. Debec (Institut Jacques Monod, UMR 7592, Paris, France)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Mad1-GFP</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>J Cell Sci. 2011 May 15;124(Pt 10):1664-71.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Mtor RNAi&#160;</td>
			<td style="width:129px;">VDRC</td>
			<td style="width:93px;">ID 110218</td>
			<td>Vienna Drosophila RNAi Center</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Materials</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:277px;">DAPI&#160;</td>
			<td style="width:129px;">Thermo</td>
			<td>D1306</td>
			<td>Stock solution must be prepared in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dumont # 5 Fine Forseps&#160;</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MBP Wide Bore Pipette Tips</td>
			<td style="width:129px;">Fisherbrand</td>
			<td>11917744</td>
			<td>Wide aperture tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#160;0.2 mL Individual Tube</td>
			<td style="width:129px;">Thermo</td>
			<td>AB-0620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml micro centrifuge tubes&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td>T3531-200EA&#160;</td>
			<td>Siliconized 2ml tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citifluor AF1</td>
			<td style="width:129px;">Citifluor</td>
			<td>AF1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dakopen&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:93px;">Z377821-1EA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum (NGS)</td>
			<td style="width:129px;">Sigma</td>
			<td>NS02L-1ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 4%</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:93px;">P6148-500G&#160;</td>
			<td>Paraformaldehyde (4%) dissolved in PBS 1X&#160;; aliquot by 600 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1X</td>
			<td style="width:129px;">Thermo</td>
			<td>14190094</td>
			<td>DPBS, no calcium, no magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% (0.2 or 0.3%) PBS-T&#160;</td>
			<td style="width:129px;"></td>
			<td></td>
			<td>PBS 1X&#160; containing 0.1% (0.2 % or 0.3%) Triton X-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100, for molecular biology,&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP booster</td>
			<td style="width:129px;">Chromotek</td>
			<td>gba488</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Mtor</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;">1/40 gift from J. Johansen, Iowa State University</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti-Nup62</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;">1/200 gift from H. Ohkura (University of Edinburgh, UK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guinea-pig anti-Ulp1</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Rabbit anti-Raf2</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DyLight conjugated series&#160;</td>
			<td style="width:129px;">Thermo</td>
			<td style="width:93px;"></td>
			<td>1/500 Donkey anti-(guinea-pig, mouse,&#160; rabbit or rat) as second antibodies</td>
		</tr>
	</tbody>
</table>

immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

The main obstacle to obtaining adequate staining of Drosophila testes is the limited penetrability of antibodies, which has been partially resolved by using squashed preparations but at the cost of inducing a restriction of the 3D analyses (for example 3D representation or measure of colocalization). The procedure permits uniform labeling and preserves the volume of all the cells of the testis. Here we have focused the illustration of the method on the 3D representation of S4-S5 spermatocytes, as they are the largest cel...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Whole-mount staining of Drosophila tissues, such as testis samples, is often problematic due to limited antibody penetration. Our fixation protocol using NP40 and heptane permits a uniform and reproducible labeling. By maintaining tissue shape while permitting deep antibody penetration, this procedure facilitates the reproducible, quantifiable acquisition of immunostaining in three dimensions, both singly and in colocalization studies.For diploid germ cell evaluation, zero to 15 hours post-eclosion, anesthetize the flies by a brief exposure to carbon dioxide, and transfer the flies to a fly pad. Place the pad under a dissecting microscope at a 10X magnification, and use forceps to remove the heads from five to 10 male flies. Transfer the decapitated fly bodies to 100 microliters of Ringer's buffer on a silicone-coated glass slide on a black background support, and use the 40X magnification and a pair of number five forceps to grasp a fly between the thorax and the abdomen.Then pull the abdomen away from the thorax and rapidly isolate the testes and adjacent tissues from the fly carcass. Once all of the testes have been collected, clean the samples of any remaining tissue and use a 200 microliter pipette tip to remove the excess buffer from around the dissected testes. Then place a drop of 4%paraformaldehyde onto the testes and quickly transfer the tissues to a tube containing two milliliters of freshly prepared 4%paraformaldehyde solution supplemented with 20%NP40 and heptane.For thorough fixation of the testes samples, shake the tube vertically for 30 seconds before incubating the testes on a rotary mixer at room temperature for 30 minutes. At the end of the incubation, transfer the tube to a tube rack and allow the testes to settle at the bottom of the tube. Remove all of the heptane and fixative solution and quickly rinse the testes three times with PBS.After the last wash, transfer the testes to a new 200 microliter tube and use a 200 microliter pipette equipped with a 10 microliter pipette tip to remove any excess buffer. Then add 200 microliters of fresh PBS-To the tube and place the testes on ice. For antibody labeling of the testes samples, replace the PBS with 200 microliters of 0.3%PBS-T for a 30-minute incubation at room temperature.After permeabilization, block any non-specific binding with 0.3%PBS-T plus 5%normal goat serum for one hour at room temperature before adding 200 microliters of the primary antibody of interest diluted in 0.3%PBS-T plus 5%normal goat serum for an overnight incubation at four degrees Celsius or room temperature with rocking. The next morning, wash the samples with three 15-minute washes with fresh 0.3%PBS-T and rocking at room temperature per wash and allow the testes to settle to the bottom of the tube by gravity. Add 200 microliters of an appropriate secondary antibody to the tube for a four-hour incubation with rocking at room temperature protected from light before washing the samples in fresh 0.3%PBS-T per wash, as demonstrated.After the 0.3%PBS-T wash, wash the samples one time in 0.2%PBS-T and one time in 0.1%PBS-T for 30 minutes per wash on the rocker, followed by a final 30-minute wash in PBS alone. After the PBS wash, label the samples with 180 microliters of a one microgram per milliliter DAPI solution in PBS for 15 minutes at room temperature protected from light, followed by three 5-minute washes in fresh PBS per wash. After the last wash, use a hydrophobic barrier pen to draw a circle on a clean slide, and use a 0.1%PBS-T primed pipette tip to transfer the testes to the slide.Aspirate any excess PBS and add 100 microliters of Citifluor AF1. Gently place a glass cover slip over the tissue, taking care to avoid trapping air bubbles, and use clear nail polish to seal the cover slip to the slide before observing the slides by confocal fluorescence microscopy. After acquiring confocal images of the samples, import the images into an appropriate image analysis software program.Then use manual segmentation to define the nucleus within each cell and determine the Pearson's correlation coefficients inside the entire volume between two fluorophores. This protocol permits the acquisition of confocal images of a sufficient quality to be treated for 3D reconstitution. For example, in these four adjacent serial sections of a stage S5 spermatocyte, a low level of Mad1 is observed at the nuclear envelope and a high-level accumulation of Mad1 can be visualized in conjunction with DAPI staining within the nucleus.in this 3D reconstituted image, the low level presence of Mad1 at the nuclear surface and high-level accumulation of Mad1 within the nucleus can be observed together with the DNA. In these 3D reconstitutions, large S5 spermatocytes stained with Nucleoporin 62 and Mad1 or Importin-beta and DAPI can be observed. In both reconstructions, the nuclear pore-associated components appear to be non-uniformly distributed over the nuclear rim as dots of irregular size.While conserving the 3D volume of the cells, this protocol also facilitates a uniform penetration of antibodies of interest into the depth of every cell, allowing quantitative analyses, such as colocalization. Because the protocol permits a reliable measurement of colocalizations from independent testes, it can also be used to compare staining in different fly genotypes. The amount of NP40 used is critical for sufficient permeabilization.If the procedure is followed closely, reproducible labeling and 3D nuclear structure preservation can be achieved. Although whole-mount staining of tissues within Drosophila can be tricky, this protocol can be easily adapted for other samples.

immunostaining-whole-mount-drosophila-testes-for-3d-confocal-analysis

Research

JoVE Journal

Biology

4.0K Views.  Université de Paris, F 75006, Paris, France. Whole-mount staining of Drosophila tissues, such as testis samples, is often problematic due to limited antibody penetration. Our fixation protocol using NP40 and heptane permits a uniform and reproducible labeling. By maintaining tissue shape while permitting deep antibody penetration, this procedure facilitates the reproducible, quantifiable acquisition of immunostaining in three dimensions, both singly and in colocalization studies.For diploid germ cell evaluation, zero to 15 hours ...