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： 在大约 70-80 毫升水中溶解 4 克 PFA。为了加快溶解过程，添加约 20 μl 的 4 N NaOH，并将溶液保存在 60 °C 的水浴中，直到 PFA 完全溶解，溶液透明。此过程大约需要 1 小时。PFA 完全溶解后，添加 10 毫升 10 倍 PBS。用水将最终体积调整至 100 毫升，冷却室温解决方案（25 °C）。为每个新实验使用新准备的解决方案。 注：获得所有动物工作的机构许可，并遵守相关动物护理准则。</li>
	</ol>
</li>
	<li>安乐死雄性老鼠。使用钳子和剪刀，从鼠标中切除睾丸。将睾丸放在PBS（或RPMI 1640）中。切除睾丸后，迅速执行以下步骤1.3-1.4，以防止RNA的退化。</li>
	<li>在一个小的培养皿上破裂睾丸，并去除 图尼卡阿尔布吉内亚。用钳子在冰上解开 PBS 中的半尼化管。</li>
	<li>使用钳子，将几个管子（几块长约5-10毫米的管子）转移到一口4井的盘子里，里面装着500毫升的CSK缓冲器+0.5%的Triton X-100在冰上，孵育6分钟。过度暴露到 CSK 缓冲区可能导致细胞中断。</li>
	<li>使用钳子，将所有管子转移到包含 4% PFA-PBS 溶液的 4 井盘中的一口井中。此步骤可以在没有立体显微镜的情况下进行预制，但可以选择地使用。在室温下孵化10分钟。同时在潜伏期开始准备12个细胞平室。</li>
	<li>使用钳子，将所有管子转移到包含 PBS 的 4 井菜的一口井中。在室温下孵化5分钟。</li>
	<li>在玻璃滑梯的背面，将所有管子转移到 30 μl 的 PBS 中（由于离子电荷，应避免滑梯顶部）。使用两个钳子的尖端，将管子撕成碎片。通过在两个钳子的尖端之间剪断管子并水平拉动钳子，将管子切成水平方向。继续此步骤约 10-20 秒。</li>
	<li>使用 P20 移液器通过管道混合悬架。</li>
	<li>将悬架转移到微中福管中，用PBS稀释至约1.3毫升。在 12 个细胞平室中每个使用 100 μl 的悬浮剂（总计 1.2 毫升）。在此步骤中，不需要确定细胞的浓度。</li>
	<li>在室温下，以 2，000 rpm 的速度将样品进行 10 分钟。细胞平移后，在室温下在实验室长凳上干滑几分钟。幻灯片允许完全干燥几分钟后，进入下一步。</li>
	<li>使用含有 PBS 的科普林罐，在室温下清洗幻灯片 5 分钟。</li>
	<li>将幻灯片转移到含有 70% 乙醇的科普林罐中，并在 RNA FISH 启动前等待至少 2 分钟。幻灯片在 70% 乙醇中稳定了几个星期， 在 4 °C 。 对于长期储存，脱水滑梯使用序列处理与80%和100%乙醇在科普林罐2分钟，空气干燥完全脱水的幻灯片。在幻灯片盒中存储在-80°C。</li>
</ol>2. 通过随机引取的 Cot-1 DNA 探针准备
<ol><li>准备以下试剂：<ol>
<li>混合缓冲：混合程序组件，准备 900 毫升杂交缓冲库存：此解决方案将用于库存探头。这种杂交缓冲库存允许在杂交前添加 10% 体积的 RNase 抑制剂溶液（核苷 Vanadyl 复合物）。<table><tbody>
<tr>
<td>元件</td>
					<td>金额（微升）</td>
					<td>最终浓度</td>
				</tr>
<tr>
<td>甲酰胺</td>
					<td>500</td>
					<td>50% （v/v）</td>
				</tr>
<tr>
<td>50% 德克斯特伦</td>
					<td>200</td>
					<td>10% （带/ v）</td>
				</tr>
<tr>
<td>20x SSC</td>
					<td>100</td>
					<td>2倍</td>
				</tr>
<tr>
<td>1% BSA</td>
					<td>100</td>
					<td>0.1%</td>
				</tr>
</tbody></table>在杂交之前，在杂交缓冲库存中加入 1/10 卷 200 mM 核苷 Vanadyl 复合物或水，以匹配最终浓度。在 -20 °C 时，这种混合物稳定一年以上。</li>
		<li>20x SSC：在大约800毫升水中溶解88.2克柠檬酸钠和175.3克纳克。使用几滴 1 M HCl 将 pH 调整到 7.0。 加入水，通过自动切割将最终体积调整到 1 L. 消毒。准备后，20倍SSC可在室温下存储。</li>
		<li>50% （w/v） 德克斯特伦： 搅拌水， 慢慢加入 50 克脱硝酸硫酸盐.在室温下保持搅拌过夜。加入水，将最终体积调整为100毫升。在室温下存放。</li>
	</ol>
</li>
	<li>将下面列出的以下成分添加到 PCR 管中。使用 PCR 机器，在 95 °C 下混合并孵育溶液 5 分钟。<table><tbody>
<tr>
<td>元件</td>
				<td align="center">每个反应的量（μl）</td>
				<td align="center">最后</td>
			</tr>
<tr>
<td>鳕鱼-1脱氧核糖核酸：1毫克/毫升</td>
				<td align="center">1</td>
				<td align="center">1 微克</td>
			</tr>
<tr>
<td>随机 9mer 引号（来自套件）</td>
				<td align="center">10</td>
				<td align="center"></td>
			</tr>
<tr>
<td>水</td>
				<td align="center">27</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>离心机短暂地放在冰上。</li>
	<li>添加以下组件。使用 PCR 机器在 37 °C 时混合孵育 30 分钟。（有关所使用的工具包的详细信息，请参阅试剂和设备表）。<table><tbody>
<tr>
<td>元件</td>
				<td align="center">每个反应的量（μl）</td>
				<td align="center">最终浓度</td>
			</tr>
<tr>
<td>从步骤 2.2 中准备的混合物</td>
				<td align="center">38</td>
				<td align="center"></td>
			</tr>
<tr>
<td>5x 核苷酸缓冲器（来自套件）</td>
				<td align="center">9.2</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Cy3-杜特普 （1 mM）</td>
				<td align="center">0.8</td>
				<td align="center">16 微米</td>
			</tr>
<tr>
<td>克莱诺 （来自套件）</td>
				<td align="center">2</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>添加2μl的停止缓冲（从套件）来停止反应。</li>
	<li>使用微针柱净化停止反应。</li>
	<li>在纯化部分添加 50 微克 （5μl） 的鱼精子 DNA（模板 Cot-1 DNA 的 50 倍） 。</li>
	<li>使用移液器测量总体积。加入 0.7 倍体积 100% 乙醇和 0.1 倍体积 3 M 醋酸钠 （pH 5.2），混合良好，离心机在最大速度 （17，000 x g） 使用微中熔体在 4 °C 下 15 分钟。</li>
	<li>去除液体，加入200微升的70%乙醇，混合好离心机5分钟，最高速度为4°C。</li>
	<li>去除多余的液体。在室温下将沉淀物干燥10-30分钟。</li>
	<li>在 20μl 的杂交缓冲库存中溶解颗粒。由于颗粒通常难以溶解，移液器反复。作为替代选项，涡流搅拌机还可用于有效溶解颗粒。探头可保持在-20°C，直到使用。</li>
</ol>3. 科特-1 RNA 鱼
<ol><li>将下面列出的组件添加到 PCR 管中。使用 PCR 机器，在 80 °C 时孵育 10 分钟，然后在 42 °C 时预接约 10-30 分钟。 保持在42°C，直到杂交。里博内库塞德瓦纳迪尔综合体是可选的;它可以被水取代。<table><tbody>
<tr>
<td>元件</td>
				<td align="center">每个反应的量（μl）</td>
				<td align="center">最后</td>
			</tr>
<tr>
<td>科特-1 DNA探针（50 ng/μl）</td>
				<td align="center">2</td>
				<td align="center">100 ng</td>
			</tr>
<tr>
<td>里博内库塞德瓦纳迪尔综合体 （200 mM）</td>
				<td align="center">2</td>
				<td align="center">20立方米</td>
			</tr>
<tr>
<td>混合缓冲区</td>
				<td align="center">添加到20</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>脱水滑入含有80%乙醇的科普林罐中2分钟，然后在2分钟内将含有100%乙醇的科普林罐中滑落。室温下，实验室长凳上完全干燥滑动。</li>
	<li>提前在 42 °C 孵化器中预热尖端盒室（充满 2-3 厘米水）。将幻灯片放在尖端盒室上。将预制探头和移液器直接直接离心到脱水滑梯上。避免制造气泡。用盖子滑动轻轻盖住幻灯片。</li>
	<li>混合6小时至夜间（14-15小时），42°C。</li>
	<li>在科普林罐中准备洗涤溶液（两个罐子，均带有 2 倍 SSC 和 50% 的表酰胺，两个罐装 2x SSC）。在42°C预热30分钟，在水浴。</li>
	<li>使用剃须刀刀片从滑梯边缘取下盖滑。避免抓取样本。在含有2x SSC和50%甲酰胺的科布林罐中清洗幻灯片，在45°C下5分钟。 重复此步骤一次。</li>
	<li>在含有2x SSC的科布林罐中清洗幻灯片5分钟，在45°C。 重复此步骤一次。</li>
	<li>在幻灯片中添加 20μl 的 DAPI 安装介质，并盖上封面滑。轻轻按盖玻璃，以去除额外的安装介质。用清洁组织点额外的安装介质。幻灯片已准备好进行显微镜评估。</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>

作者无可透露。

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>
		<col />
		<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

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。

幻灯片制备方法，以保存睾丸细菌细胞的三维染色质结构

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

slide-preparation-method-to-preserve-three-dimensional-chromatin

Research

JoVE Journal

Biology

7.3K 次观看.  Cincinnati Children's Hospital Medical Center. 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。

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

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

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.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

简单和高效的睾丸细胞悬液的制备技术

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

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.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

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 ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

无源清清除小鼠卵巢血管构筑的三维重建

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

使用三维共培养系统NICO-1评估卵巢癌干细胞样细胞的血管遗传特性

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...