Name: slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells
Uploaded: 2014-01-10T14:15:00
Description: Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

我感谢珍妮 T. 李监督这个项目时， 我在李实验室和泰勒布罗林编辑手稿。这项工作得到了迪姆斯基金会3月颁发的巴兹尔·奥康纳初学者奖和NIH赠款GM098605的支持。

1.3D 幻灯片准备
<ol><li>准备以下试剂：<ol>
<li>CSK 缓冲器：100 mM 纳克、300 米蔗糖、10 米管道、3 毫米 MgCl2。将pH调整为6.8，并配备1 M NAOH。将 CSK 缓冲区存储在 4 °C。</li>
		<li>CSK 缓冲区，带 0.5% 特里顿 X-100：添加 200 μl 的特里顿 X-100 至 40 毫升 CSK 缓冲器。使用磁搅拌器混合溶液，直到 Triton X-100 完全溶解。将 CSK 缓冲区存储在 4 °C 的 0.5% 特里顿 X-100 中。</li>
		<li>4% 甲醛 （PFA）– PBS， pH 7.4...

<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>

在 3D 幻灯片准备中，渗透步骤先于固定步骤。这些步骤直接在半细管上执行，从而保持三维染色质结构。保存RNA/DNA FISH染色质结构的另一种选择是同时执行渗透步骤和固定步骤。这种替代技术已在母体生殖细胞和小鼠预植胚胎7，8中进行。但是，如果固定步骤先于渗透步骤，则可能导致高细胞质背景。因此，RNA和DNA FISH滑动优化的关键决定因素取决于渗透顺序和固定步骤。渗透步骤的持续...

在睾丸中，生殖细胞通过肌瘤从双体精氨酸中分离成成熟的、生物体精子。在此过程中，不断动态改造生殖细胞的核染色质结构。表面扩散通常用于个体精子细胞的细胞学检查。表面扩散的一种流行方法采用低血压治疗，通过这种疗法，个体精子细胞被扩散和扁平化。这些条件最适合详细分析美色体。使用这种方法很容易观察到染色体特征，如突触状态和重组 foci。然而，低音治疗破坏亚核染色质结构，因此该技术不适合核结构分析。因此，设计了一种改进的方法来保存睾丸生殖细胞的三维染色质结构。此方法被称为三维 （3D） 滑动法 （图1）。3D滑动方法使核中新生RNA定位的检测成为可能，因为它最初经过优化，通过RNA荧光在原地杂交（FISH）2，3中检查睾丸细菌细胞分化过程中的基因表达和色度状态。这种3D方法也适用于免疫荧光、DNA和RNA FISH的组合。此外，这种方法还有助于发现后美化性染色质（PMSC），这是在后美化精子2中发现的性染色体的无声隔间。
3D 滑动方法最初是通过用于核染色的滑动制备的两个基本步骤的组合进行优化的：用于修复核材料的固定步骤和旨在去除细胞质材料的渗透步骤，以提高染色试剂（如抗体和 FISH 探头）的可访问性。正如另一本出版物3中先前所述，已经确定渗透步骤必须在固定步骤之前进行，以便在低细胞质背景下获得最佳结果。在这种3D滑动方法中，渗透步骤和随后的固定步骤直接在半细管上执行，然后在细胞固定到滑梯上之前，用钳子将生殖细胞与钳子进行机械分离。另一个实验室4开发了一种精子生殖细胞RNA FISH的替代方法。在此方法中，与 3D 方法一致，渗透步骤必须在固定步骤之前进行，以便获得 RNA FISH 的最佳结果。
下列协议描述了 3D 滑动方法，并提供了可能应用 Cot-1 RNA FISH 检测核新生RNA的示例。Cot-1 DNA由基因组中的重复元素组成。在这里，一个Cot-1DNA探针被杂交到新生成绩单的直肠和UTR，从而检测核5，6的转录活跃区域。3D 幻灯片用途广泛，可应用于免疫荧光、DNA 和 RNA FISH 技术的组合，以便详细检查染色质架构之间的空间关系。

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<colgroup>
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cot-1 DNA&#160;</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stratagene Prime-It Fluor Fluorescence Labeling Kit</td>
			<td style="width:147px;">Agilent</td>
			<td align="right" style="width:119px;">
			300380
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herring sperm DNA Solution</td>
			<td style="width:147px;">Invitrogen</td>
			<td>15634-017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 200 proof (absolute)</td>
			<td>Pharmco-AAPER</td>
			<td align="right">111000200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Formamide deionized</td>
			<td>American Bioanalytical</td>
			<td>AB00600-00500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate tribasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C8532</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:147px;">Lifeblood Medical</td>
			<td style="width:119px;">77110-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt from Leuconostoc spp.</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D6001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">A10491-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">M0250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">76240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">221465</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P4417</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Mounting Medium with DAPI</td>
			<td style="width:147px;">Vector Laboratories</td>
			<td style="width:119px;">H1200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonucleoside-vanadyl complex</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">S1402S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illustra MicroSpin G-50 Columns</td>
			<td>GE Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge</td>
			<td style="width:147px;">Eppendorf</td>
			<td align="right" style="width:119px;">5424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">300-050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge tubes</td>
			<td style="width:147px;">Bioexpress</td>
			<td style="width:119px;">C-3262-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cytospin 3</td>
			<td style="width:147px;">Shandon</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Coplin jar</td>
			<td style="width:147px;">Fisher</td>
			<td style="width:119px;">08-815</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dish</td>
			<td>Fisher</td>
			<td style="width:119px;">12-566-301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass&#160;</td>
			<td>Fisher</td>
			<td style="width:119px;">12-544-G</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Air incubator</td>
			<td style="width:147px;">Revolutionary Science</td>
			<td style="width:119px;">RS-IF-203</td>
		</tr>
	</tbody>
</table>

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

在睾丸生殖细胞分化过程中，核染色质的结构动态变化。下列描述了一种旨在保存小鼠睾丸生殖细胞的三维染色质排列的方法：此方法被称为三维 （3D） 滑动方法。在这种方法中，睾丸管直接处理与渗透步骤，去除细胞质材料，然后固定步骤，修复核材料。然后，图布分离，细胞悬浮是细胞，细胞粘附在幻灯片上。该方法提高了对亚核结构检测的敏感性，适用于 原位 杂交（FISH）的免疫荧光、DNA和RNA荧光，以及这些检测方法的组合。作为 3D 幻灯片方法可能应用的示例，可显示 Cot-1 RNA FISH 检测新生的RNA。3D滑动方法将促进睾丸生殖细胞分化过程中色度结构、DNA和RNA之间的空间关系的详细检查。

3D 幻灯片的代表性结果显示在 图 2 中。在这个实验中，使用抗CBX1和γH2AX抗体检测新生转录和免疫染色。为了结合RNA FISH和免疫染色，首先进行免疫染色，然后是RNA FISH，如3所述。CBX1是一种异色素蛋白，其局部化为外周异色素和无声性染色体，称为XY体（或性体）。γH2AX是一种磷化形式的高石变种H2AX和XY身体的局部。在这张照片中，Cot-1信号积聚在欧色学区域，但被排除在外周...

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

幻灯片制备方法，以保存睾丸细菌细胞的三维染色质结构

这里的材料描述了一种为保存睾丸生殖细胞的三维染色质结构而开发的方法。这被称为三维（3D）滑动方法。该方法提高了对亚核结构检测的灵敏度，适用于 原位 杂交（FISH）中的免疫荧光、DNA和RNA荧光。

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

Research

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芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

科研

生物学

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

嗨 - C：一个方法研究基因组的三维架构。

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Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

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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 ...

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