I thank Jeannie T. Lee for supervision of this project when I was in the Lee laboratory and Tyler Broering for editing the manuscript. This work was supported by the Basil O&#8217;Connor Starter Scholar Award from the March of Dimes Foundation and the NIH Grant GM098605.

1. 3D Slide Preparation
<ol>
	<li>Prepare the following reagents:
	<ol>
		<li>CSK buffer: 100 mM NaCl, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl2. Adjust pH to 6.8 with 1 M NaOH. Store the CSK buffer at 4 &#176;C.</li>
		<li>CSK buffer with 0.5% Triton X-100: Add 200 &#956;l of Triton X-100 to 40 ml of CSK buffer. Mix the solution using magnetic stirrer until Triton X-100 is completely dissolved. Store the CSK buffer with 0.5% Triton X-100 at 4 &#176;C.</li>
		<li>4% Paraformaldehyde (PFA)&#8211;PBS, pH 7.4: Dissolve 4 g of PFA in approximately 70-80 ml of water. In order to expedite the dissolving process, add approximately 20 &#956;l of 4 N NaOH and keep the solution in a water bath at 60 &#176;C until the PFA has completely dissolved and the solution is transparent. This process takes approximately 1 hr. After PFA has completely dissolved, add 10 ml of 10x PBS. Adjust the final volume to 100 ml with water and cool down the solution to room temperature (25 &#176;C). Use freshly prepared solution for each new experiment. 
		Note: Obtain institutional permission for all animal work and adhere to relevant animal care guidelines.</li>
	</ol>
	</li>
	<li>Euthanize male mice. Using forceps and scissors, excise the testis from the mouse. Place the testis in PBS (or RPMI 1640). After the removal of testis, quickly carry out the following steps 1.3-1.4 in order to prevent the degradation of RNA.</li>
	<li>Rupture the testis on a small Petri dish and remove the tunica albuginea. Unravel the seminiferous tubules in PBS on ice using forceps.</li>
	<li>Using forceps, transfer several tubules (several pieces of tubules approximately 5-10 mm in length) into one well of a 4-well dish containing 500 ml of CSK buffer + 0.5% Triton X-100 on ice, and incubate for 6 min. Overexposure to CSK buffer may result in the disruption of cells.</li>
	<li>Using forceps, transfer all the tubules to one well of a 4-well dish containing 4% PFA-PBS solution. This step can be preformed without a stereomicroscope, but optionally it can be used. Incubate for 10 min at room temperature. Simultaneously begin preparing 12 cytospin chambers during the incubation periods.</li>
	<li>Using forceps, transfer all the tubules to one well of a 4-well dish containing PBS. Incubate for 5 min at room temperature.</li>
	<li>On the backside of a glass slide, transfer all the tubules into 30 &#956;l of PBS (the top of the slide should be avoided due to cationic charge). Using the tips of two forceps, tear the tubules to pieces. Chop the tubules in a horizontal direction by clipping tubules between tips of two forceps and pulling the forceps horizontally. Continue with this step for approximately 10-20 sec.</li>
	<li>Mix the suspension by pipetting using a P20 pipette.</li>
	<li>Transfer the suspension to a microcentrifuge tube, dilute with PBS to approximately 1.3 ml. Apply 100 &#956;l of suspension to each of the 12 cytospin chambers (1.2 ml total). At this step, the concentration of cells does not need to be determined.</li>
	<li>Cytospin the samples at 2,000 rpm for 10 min at room temperature. After cytospinning, dry slides on lab bench for a few minutes at room temperature. After slides have been allowed to dry completely for few minutes, go to the next step.</li>
	<li>Using a Coplin jar containing PBS, wash the slides for 5 min at room temperature.</li>
	<li>Transfer slides to a Coplin jar containing 70% ethanol and wait at least 2 min prior to initiation of RNA FISH. Slides are stable for a few weeks in 70% ethanol at 4 &#176;C. For long-term storage, dehydrate slides using serial treatment with 80% and 100% ethanol in Coplin jars for 2 min each and air-dry completely dehydrate the slides. Store at -80 &#176;C in a slide box.</li>
</ol>
2. Cot-1 DNA Probe Preparation by Random Priming
<ol>
	<li>Prepare the following reagents:
	<ol>
		<li>Hybridization buffer: Mix the proceeding components to prepare 900 ml of hybridization buffer stock; this solution will be used to stock probes. This hybridization buffer stock allows for the addition of 10% volume of RNase inhibitor solution (Ribonucleoside Vanadyl Complex) prior to hybridization.
		<table>
			<tbody>
				<tr>
					<td>Component</td>
					<td>Amount (&#956;l)</td>
					<td>Final concentration</td>
				</tr>
				<tr>
					<td>Formamide</td>
					<td>500</td>
					<td>50% (v/v)</td>
				</tr>
				<tr>
					<td>50% Dextran</td>
					<td>200</td>
					<td>10% (w/v)</td>
				</tr>
				<tr>
					<td>20x SSC</td>
					<td>100</td>
					<td>2x</td>
				</tr>
				<tr>
					<td>1% BSA</td>
					<td>100</td>
					<td>0.1%</td>
				</tr>
			</tbody>
		</table>
		Prior to hybridization, add &#8530; vol of 200 mM Ribonucleoside Vanadyl Complex or water to the hybridization buffer stock to match the final concentration. At -20 &#176;C, this mixture is stable for more than a year.</li>
		<li>20x&#160;SSC: Dissolve 88.2 g of sodium citrate and 175.3 g of NaCl in approximately 800 ml of water. Adjust the pH to 7.0 using a few drops of 1 M HCl. Add water to adjust the final volume to 1 L. Sterilize by autoclaving. After preparation, 20x&#160;SSC can be stored at room temperature.</li>
		<li>50% (w/v) Dextran: Stir water and slowly add 50 g of dextran sulfate. Keep stirring overnight at room temperature. Add water to adjust final volume to 100 ml. Store at room temperature.</li>
	</ol>
	</li>
	<li>Add the following constituents listed below to a PCR tube. Using a PCR machine, mix and incubate the solution for 5 min at 95 &#176;C.
	<table>
		<tbody>
			<tr>
				<td>Component</td>
				<td align="center">Amount per reaction (&#956;l)</td>
				<td align="center">Final</td>
			</tr>
			<tr>
				<td>Cot-1 DNA: 1 mg/ml</td>
				<td align="center">1</td>
				<td align="center">1 &#956;g</td>
			</tr>
			<tr>
				<td>Random 9mer primer (from kit)</td>
				<td align="center">10</td>
				<td align="center"></td>
			</tr>
			<tr>
				<td>Water</td>
				<td align="center">27</td>
				<td align="center"></td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Centrifuge briefly and place on ice.</li>
	<li>Add the following components. Mix and incubate for 30 min at 37 &#176;C using a PCR machine. (For details of the kit used see the table of reagents and equipment).
	<table>
		<tbody>
			<tr>
				<td>Component</td>
				<td align="center">Amount per reaction (&#956;l)</td>
				<td align="center">Final concentration</td>
			</tr>
			<tr>
				<td>Mixture prepared from step 2.2</td>
				<td align="center">38</td>
				<td align="center"></td>
			</tr>
			<tr>
				<td>5x nucleotide buffer (from kit)</td>
				<td align="center">9.2</td>
				<td align="center"></td>
			</tr>
			<tr>
				<td>Cy3-dUTP (1 mM)</td>
				<td align="center">0.8</td>
				<td align="center">16 &#956;M</td>
			</tr>
			<tr>
				<td>Klenow (from kit)</td>
				<td align="center">2</td>
				<td align="center"></td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Add 2 &#956;l of stop buffer (from kit) to discontinue the reaction.</li>
	<li>Purify the stopped reaction using microspin columns.</li>
	<li>Add 50 &#956;g (5 &#956;l) of herring sperm DNA (50 fold excess of template Cot-1 DNA) to the purified fraction.</li>
	<li>Measure the total volume using a pipette. Add 0.7x volume of 100% ethanol and 0.1x volume of 3 M sodium acetate (pH 5.2), mix well, and centrifuge for 15 min at maximum speed (17,000 x g) using a microcentrifuge at 4 &#176;C.</li>
	<li>Remove the liquid, and add 200 &#956;l of 70% ethanol, mix well and centrifuge for 5 min at maximum speed at 4 &#176;C.</li>
	<li>Remove the excess liquid. Dry the precipitate for 10-30 min at room temperature.</li>
	<li>Dissolve the pellet in 20 &#956;l of hybridization buffer stock. Because the pellet is often difficult to dissolve, pipette repeatedly. As an alternate option, a vortex mixer can also be used to efficiently dissolve the pellet. Probes can be kept at -20 &#176;C until use.</li>
</ol>
3. Cot-1 RNA FISH
<ol>
	<li>Add the components listed below to a PCR tube. Using a PCR machine, incubate for 10 min at 80 &#176;C, followed by a preannealing step of approximately 10-30 min at 42 &#176;C. Keep at 42 &#176;C until hybridization. Ribonucleoside Vanadyl Complex is optional; it can be replaced with water.
	<table>
		<tbody>
			<tr>
				<td>Component</td>
				<td align="center">Amount per reaction (&#956;l)</td>
				<td align="center">Final</td>
			</tr>
			<tr>
				<td>Cot-1 DNA probe (50 ng/&#956;l)</td>
				<td align="center">2</td>
				<td align="center">100 ng</td>
			</tr>
			<tr>
				<td>Ribonucleoside Vanadyl Complex (200 mM)</td>
				<td align="center">2</td>
				<td align="center">20 mM</td>
			</tr>
			<tr>
				<td>Hybridization buffer</td>
				<td align="center">added to 20</td>
				<td align="center"></td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Dehydrate slides in a Coplin jar containing 80% ethanol for 2 min, followed by a Coplin jar containing 100% ethanol for 2 min. Dry slides completely on lab bench at room temperature.</li>
	<li>Preheat a tip box chamber (filled with 2-3 cm of water) in a 42 &#176;C incubator in advance. Place the slide on the tip box chamber. Briefly centrifuge the preannealed probes and pipette directly onto the dehydrated slides. Avoid making bubbles. Gently cover the slide with a cover slip.</li>
	<li>Hybridize for 6 hr to overnight (14-15 hr) at 42 &#176;C.</li>
	<li>Prepare wash solutions in Coplin jars (two jars with both 2x SSC and 50% formamide, and two jars with 2x SSC). Preheat at 42 &#176;C for 30 min in a water bath.</li>
	<li>Use a razor blade to remove the cover slip from the edge of the slide. Avoid scratching samples. Wash slides in a Coplin jar containing 2x SSC and 50% formamide for 5 min at 45 &#176;C. Repeat this step once.</li>
	<li>Wash slides in a Coplin jar containing 2x SSC for 5 min at 45 &#176;C. Repeat this step once.</li>
	<li>Add 20 &#956;l of DAPI-mounting media to the slides, and cover with a cover slip. Gently press cover glass to remove extra mounting media. Blot extra mounting media with cleaning tissue. The slides are ready for microscopy evaluation.</li>
</ol>

<ol>
	<li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Namekawa, S. H., 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=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr Biol. 16, 660-667 (2006).</li><li>Namekawa, S. H., Lee, 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=Detection+of+nascent+RNA,+single-copy+DNA+and+protein+localization+by+immunoFISH+in+mouse+germ+cells+and+preimplantation+embryos.">Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.</a> Nat Protoc. 6, 270-284 (2011).</li><li>Mahadevaiah, S. K., Costa, Y., Turner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA+FISH+to+study+gene+expression+during+mammalian+meiosis.">Using RNA FISH to study gene expression during mammalian meiosis.</a> Methods in molecular biology. 558, 433-444 (2009).</li><li>Hall, L. L., 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=An+ectopic+human+XIST+gene+can+induce+chromosome+inactivation+in+postdifferentiation+human+HT-1080+cells.">An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.</a> Proc Natl Acad Sci U S A. 99, 8677-8682 (2002).</li><li>Huynh, K. D., Lee, 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=Inheritance+of+a+pre-inactivated+paternal+X+chromosome+in+early+mouse+embryos.">Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos.</a> Nature. 426, 857-862 (2003).</li><li>Namekawa, S. H., VandeBerg, J. L., McCarrey, J. R., Lee, 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=Sex+chromosome+silencing+in+the+marsupial+male+germ+line.">Sex chromosome silencing in the marsupial male germ line.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9730-9735 (2007).</li><li>Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R., Lee, 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=Two-step+imprinted+X+inactivation:+repeat+versus+genic+silencing+in+the+mouse.">Two-step imprinted X inactivation: repeat versus genic silencing in the mouse.</a> Mol Cell Biol. 30, 3187-3205 (2010).</li><li>Ichijima, Y., 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=MDC1+directs+chromosome-wide+silencing+of+the+sex+chromosomes+in+male+germ+cells.">MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.</a> Genes Dev. 25, 959-971 (2011).</li><li>Sin, H. 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=RNF8+regulates+active+epigenetic+modifications+and+escape+gene+activation+from+inactive+sex+chromosomes+in+post-meiotic+spermatids.">RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids.</a> Genes Dev. 26, 2737-2748 (2012).</li></ol>

The author has nothing to disclose.

In 3D slide preparation, the permeabilization step precedes the fixation step. These steps are performed directly on seminiferous tubules, thereby preserving the three-dimensional chromatin structure. An alternate option to preserving chromatin structure for RNA/DNA FISH is to perform the permeabilization step and the fixation step simultaneously. This alternate technique has been performed in marsupial germ cells and in mouse preimplantation embryos7,8. However, if the fixation step precedes the permeabilization step, it may give rise to high cytoplasmic background. Therefore, the critical determinant of slide optimization for RNA and DNA FISH is dependent upon the order of the permeabilization and the fixation steps. The duration of the permeabilization step is also an important factor for the optimization of slide preparation. Six minutes is the general condition for this method. However, it can be adjusted depending on variation of the materials used.
The preservation of the three-dimensional chromatin structure is a feature of the 3D slide that distinctly separates it from surface spread slides. This advantage differs greatly from that of chromosome spread preparation. The 3D slide method preserves three-dimensional chromatin structure, but requires data acquisition with multiple z-sections to encompass an entire nucleus. A single z-section cannot capture all signals of a nucleus. Thus, the necessity for multiple z-sections is a major limitation to this method. To capture a nucleus in a single z-section, surface spreads have an advantage. Therefore, the 3D method and the surface spread preparation have distinct advantages&#160;and mutually compensate the features of each other for the study of meiosis and spermatogenesis.
The 3D slide method can be applied to all cell types found within the testicles, including spermatogonia, round spermatids, and Sertoli cells. This method has also been applied to examine the chromatin compaction of the sex chromosomes at the onset of meiotic sex chromosome inactivation, and to examine epigenetic modifications on PMSC10. In terms of future applications, this method may be applied to other tissues or cells. If so, it is recommended that the permeabilization step precede the fixation step. Alternatively, the permeabilization step and the fixation step can be performed simultaneously.

In testes, germ cells differentiate from diploid spermatogonia through meiosis into mature, haploid spermatozoa. During this process, the nuclear chromatin structure of germ cells is continuously and dynamically remodeled. Surface spreads are commonly used for the cytological examination of individual spermatogenic cells. A prevailing method of surface spreads employs hypotonic treatment by which individual spermatogenic cells are spread and flattened1. These conditions are optimal for detailed analysis of meiotic chromosomes. Chromosomal features such as synaptic status and recombination foci are easily observed using this method. However, the hypotonic treatment disrupts subnuclear chromatin architecture, thus this technique is not suitable for structural analysis of nuclei. Consequently, an improved method has been designed to preserve the three-dimensional chromatin structure of testicular germ cells. This method has been termed as the three-dimensional (3D) slide method (Figure 1). The 3D slide method has enabled the detection of nascent RNA localization in nuclei because it was initially optimized to examine gene expression and chromatin states during testicular germ cell differentiation by RNA fluorescence in situ hybridization (FISH)2,3. This 3D method is also applicable to the combination of immunofluorescence, DNA, and RNA FISH. Additionally, this method has aided in the discovery of postmeiotic sex chromatin (PMSC), a silent compartment of the sex chromosomes found in postmeiotic spermatids2.
The 3D slide method was originally optimized through the combination of two essential steps of slide preparation commonly used for nuclear staining: the fixation step designed to fix nuclear materials&#160;and the permeabilization step intended to remove cytoplasmic materials in order to improve the accessibility of staining reagents, such as antibodies and FISH probes. As previously described in another publication3, it has been determined that the permeabilization step must precede the fixation step in order to obtain optimal results with low cytoplasmic background. In this 3D slide method, the permeabilization step and subsequent fixation step are performed directly on seminiferous tubules&#160;and are followed by the mechanical dissociation of germ cells with forceps prior to cytospinning onto slides. An alternative method for RNA FISH of spermatogenic cells was developed in another laboratory4. In this method, consistent with the 3D method, the permeabilization step must precede the fixation step in order to obtain optimal results of RNA FISH.
The following protocol describes the 3D slide method and provides an example of a possible application, Cot-1 RNA FISH for the detection of nuclear nascent RNAs. Cot-1 DNA consists of repetitive elements in the genome. Here, a Cot-1 DNA probe is hybridized to an intron and UTR of the nascent transcripts, thereby detecting the transcriptionally active regions in nucleus5,6. 3D slides are versatile and can be applied to a combination of immunofluorescence, DNA and RNA FISH techniques in order to obtain detailed examination of spatial relationships between chromatin architecture.

<table><tbody><tr><td>Cot-1 DNA&amp;nbsp;</td><td>Invitrogen</td><td>18440-016</td><td></td></tr><tr><td>Stratagene Prime-It Fluor Fluorescence Labeling Kit</td><td>Agilent</td><td>300380</td><td></td></tr><tr><td>Herring sperm DNA Solution</td><td>Invitrogen</td><td>15634-017</td><td></td></tr><tr><td>Ethanol. 200 proof (absolute)</td><td>Pharmco-aaper</td><td>111000200</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Formamide deionized</td><td>American Bioanalytical</td><td>AB00600-00500</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma-Aldrich</td><td>C8532</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Lifeblood Medical</td><td>77110-100</td><td></td></tr><tr><td>Dextran sulfate sodium salt from Leuconostoc spp.</td><td>Sigma-Aldrich</td><td>D6001</td><td></td></tr><tr><td>RPMI 1640</td><td>Invitrogen</td><td>A10491-01</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>76240</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>VECTASHIELD Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H1200</td><td></td></tr><tr><td>Ribonucleoside-vanadyl complex</td><td>New England Biolabs</td><td>S1402S</td><td></td></tr><tr><td>Illustra MicroSpin G-50 Columns</td><td>GE Healthcare</td><td>27-5330-01</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>300-050</td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Bioexpress</td><td>C-3262-1</td><td></td></tr><tr><td>Cytospin 3</td><td>Shandon</td><td></td><td></td></tr><tr><td>Coplin Jar</td><td>Fisher</td><td>08-815</td><td></td></tr><tr><td>Superfrost /Plus Microscope Slides</td><td>Fisher</td><td>12-550-15</td><td></td></tr><tr><td>4-well dish</td><td>Fisher</td><td>12-566-301</td><td></td></tr><tr><td>Cover glass&amp;nbsp;</td><td>Fisher</td><td>12-544-G</td><td></td></tr><tr><td>Air incubator</td><td>Revolutionary Science</td><td>RS-IF-203</td><td></td></tr></tbody></table>

slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

A representative result of a 3D slide is shown in Figure 2. In this experiment, Cot-1 RNA FISH was performed to detect nascent transcription together with immunostaining using anti CBX1 and &#947;H2AX antibodies. To combine RNA FISH and immunostaining, immunostaining was performed first, followed by RNA FISH as described in3. CBX1 is a heterochromatin protein that localizes to pericentromeric heterochromatin and silent sex chromosomes in meiosis called an XY body (or sex body). &#947;H2AX is a phosphorylated form of histone variant H2AX and localizes on the XY body. In this picture, Cot-1 signals accumulate on euchromatic regions, but are excluded from pericentromeric heterochromatin and the XY body.
During surface spread preparation, treatment with hypotonic solution swells nuclei and physically extends subnuclear structures. This prevailing method for meiotic study is best suited for capturing all meiotic chromosomes in a single z-plane1. To clarify the difference between the 3D slide method and surface spreads after hypotonic treatment, representative pictures of pachytene spermatocytes of each experiment are shown using the same magnification using an epifluorescent microscope (Figures 2A and 2B). In surface spreads treated with hypotonic solution, the three-dimensional chromatin structure is disrupted and all meiotic chromosome axes can be captured within a single z-plane. 3D slide preparation, however, preserves the intact chromatin structure.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50819/50819fig1highres.jpg" src="/files/ftp_upload/50819/50819fig1.jpg" /> 
Figure 1. Schematic of 3D slide method. The 3D slide method can preserve the three-dimensional chromatin structure. Due to the three-dimensional nature of the slide, data acquisition with multiple z-section is required to cover a nucleus.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50819/50819fig2highres.jpg" src="/files/ftp_upload/50819/50819fig2.jpg" /> 
Figure 2. Comparison between the 3D slide method and surface spreads after hypotonic treatment at the same magnification. (A) Using the 3D slide, Cot-1 RNA FISH (cy3) and immunostaining with anti CBX1 (fitc) and &#947;H2AX (cy5) antibodies are performed. A pachytene spermatocyte with a single z-section is shown. (B) Using the surface spreads, immunostaining with anti SCP3 and &#947;H2AX antibodies are performed. A pachytene spermatocyte is shown. Circles: XY body. Bars: 10 &#956;m.

Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

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7.3K Views.  Cincinnati Children's Hospital Medical Center. The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Video: Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Genetics

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Developmental Biology

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

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.

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.

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

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

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Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

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

Imaging Centrosomes in Fly Testes

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes