The authors thank all previous and present members of the department of Reproduction and Development of the Erasmus MC for helpful and stimulating discussions during the past years. We also want to thank the three anonymous reviewers for providing helpful feedback. Work in the Gribnau lab is supported by funding from the Dutch research council (NWO-VICI) and an ERC starting grant.

1，分化的女性胚胎干细胞诱导为诱导X染色体失活注：被女小鼠胚胎干细胞（可根据要求）​​上涂有小鼠胚胎成纤维细胞（MEF中）糊化培养皿标准ES细胞的条件下生长。在这里，我们假设读者熟悉标准的细胞培养技术39-41。为了诱导分化，在T25菜生长的ES细胞会从MEF中被分离，并在分化培养基将被镀金。 <ol><li>从ES细胞培养物中移除ES培养基，并用细胞培养PBS洗涤两次。 </li><li>加1.5毫升胰蛋白酶-EDTA（预热至37℃），并培养细胞，在37℃7分钟。经过3.5分钟，摇盘轻轻向上突破的殖民地。 </li><li>通过添加3.5毫升分化培养基对细胞灭活胰蛋白酶-EDTA。上下吸液管的细胞，以获得单细胞溶液中，并收集在一个15毫升管。自旋为5分钟在200 XG，并收集细胞于10ml分化培养基中。 </li><li>添加细胞悬浮液到一个非糊化的培养皿中，并孵育1小时，在细胞培养箱中。注意：MEFs细胞将能够附着到非糊化的培养皿中，而ES细胞将保持在悬浮液中。 </li><li>在此期间，添加无菌盖玻片（24×24毫米）到6孔板中，添加0.2％的明胶，和孵育最少5分钟，在室温下进行。 </li><li> 1小时后，收集的预镀的ES细胞培养物的上清液，其中将主要由非粘附的ES细胞。板ES细胞于含有盖玻片的6孔​​板。注意：使用⅙ 日汇合T25菜6口井6孔板的。这将允许一个融合后的分化以及分化3天。每天更换培养基。对于较长的微分时间的课程，板预镀ES细胞在细菌的菜肴分化培养基，并incub吃的小区摇床整合到细胞培养孵化器。这将允许细胞形成胚状体，它可在固定前1-2天接种到盖玻片。 </li></ol>分化的胚胎干细胞用于后续的DNA-RNA FISH分析2。固定<ol><li>从分化培养移除分化培养基，并用细胞培养的PBS洗两次。注：添加的PBS小心，以防止被冲走的细胞了。 </li><li>除去细胞培养的PBS，固定细胞，加入4％多聚甲醛（PFA）/ PBS中，并孵育在室温下10分钟。注意：PFA是有毒的，可引起显著的健康风险被吸入或接触到皮肤或眼睛时，时。此外，PFA是致癌物质。 </li><li>除去4％PFA / PBS中，并洗涤盖玻片3次，用70％的乙醇。注意：正确的洗涤非常重要，因为煤灰的痕迹将与随后的鱼步骤干涉。盖玻片可以在-20℃几个月前至F被存储在70％乙醇原位杂交分析。 </li></ol> 3，标记DNA探针的FISH实验注：获得目的基因（最小长度&gt; 2 kb的RNA的鱼，或&gt; 20 kb的DNA的FISH），或BAC覆盖目的基因的DNA片段。用于RNA检测，相对于DNA的检测更小的探针可以是足够的，因为最可能的转录将导致多个转录物，它可以被同时检测到。对于单拷贝DNA链上的强烈信号，较长的探针是必需的，以便能够检测结合的半抗原有足够数量。优选地，非转录的基因组区域被选择为设计上的DNA探针。此外，使用较小的探针来检测的RNA时，它会被防止被检测的结合DNA-RNA FISH方法的基因组位点从该RNA的转录，尽管这不能完全排除。来检测鼠标Xist的 ，一个5.5kb的cDNA探针使用。对于DETX染色体上的挠度，几个BAC探针都可以使用。良好的结果已获得的BAC RP23-100E1和BAC CT7-474E4，分别位于邻近的Xist位点的混合物。根据感兴趣的基因或染色体上，多个不同的探针可能需要取得最佳的效果。只要有材料，将用于RNA-FISH工作，用无菌过滤嘴，无RNA酶H 2 O和工作在一个干净的环境。 <ol><li>取1的DNA微克和稀的H 2 O的在16微升的总体积。加入4微升地高辛或生物素标记液（见试剂），涡旋混匀，于16℃下90分钟。注：生物素或地高辛标记的核苷酸将通过缺口平移纳入。 </li><li>在此期间，准备G50柱以除去非集成核苷酸。取2毫升的注射器，取出模具，并添加消毒棉入注射器。加入G50球在溶液吨Ø填充注射器，将注射器在15毫升管和离心642×g离心1分钟。注意：大约80％的注射器现在应该充满G50。当重复G50少存在。 </li><li> 90分钟后，加入40微升H 2 O到缺口平移反应混合物，并添加反应到G50柱。添加一个空管栏下，并在离心642×g离心2分钟。通过在一个反应​​管中收集的流动。 </li><li>通过用移液管测量流量的容积，并添加H 2 O至达到200μl的总体积。通过加入13.3微升的鲑精DNA（10毫克/毫升），13.3微升酵母tRNA（10毫克/毫升），26.7微升小鼠摇篮-1 DNA（1毫克/毫升）和28微升的2M乙酸钠，pH值沉淀出流5.6。通过涡旋混合，并加入533微升冰冷的100％乙醇。摇管中，离心16,200×g离心30分钟，在4℃下</li><li>去除上清，并用70％乙醇洗两次沉淀。离心机在16,200×g离心5分钟，4℃，空气干燥沉淀。添加50微升50％+的杂交溶液中，在37℃下进行30分钟，以促进溶解。注意：标记探针可存储数年，在-20℃下注意：50 +杂交溶液中含有甲酰胺，它是有毒的，会刺激皮肤，眼睛和呼吸系统，也是致畸。 </li></ol> 4，通透，前处理固定细胞，并用于杂交组合DNA-RNA鱼<ol><li>再水化的固定的细胞在盖玻片上，通过除去70％的乙醇和另外的细胞培养物的PBS于6孔板中。孵育5分钟。在重复共三次。 </li><li>除去细胞培养PBS中，并加入0.2％的胃蛋白酶于6孔板中，可渗透细胞。孵育6孔板中在水浴中于37℃持续4分钟。除去胃蛋白酶，和失活与无RNA酶的H 2 O。 </li><li>定位后的细胞用4％PFA / PBS中温育5分钟，在室温下进行。之后fixati上，用细胞培养的PBS洗两次，每次5分钟。 </li><li>通过用70％乙醇温育3分钟，90％乙醇中3分钟，100％乙醇中3分钟，脱水的细胞。转让盖玻片的6孔​​板上，并空气干燥盖玻片几分钟。 </li><li>年龄细胞孵育盖玻片，在65℃下进行1小时的热板。 </li><li>加在一起，每盖玻片进行分析预杂交用鼠标COT-1的DNA FISH探针，25微升50 +杂交液，0.5μL鼠标COT-1的DNA，1微升生物素标记的Xist探头和1μl地高辛的标记的BAC探针检测X染色体上。通过涡旋混合，并在变性99℃5分钟。立即在37℃保持45分钟，以允许摇篮-1的DNA杂交，重复存在于探针的序列。 </li><li>老化后，发现将50μl变性缓冲液（由70％formamide/2x SSC/10 mM磷酸盐缓冲液），在载玻片上，并置于盖玻片上顶用细胞面临towarDS变性缓冲。孵育在65℃持续2分钟。 </li><li>通过温育在冰冷的70％乙醇5分钟加盖玻片回6孔板中，并定位后的细胞。 </li><li>通过随后的温育在70％乙醇脱水的细胞为3分钟，90％乙醇中3分钟，100％乙醇中3分钟。从6孔培养板除去盖玻片，并让空气干燥几分钟。 </li><li>发现在载玻片上预杂交探针，并孵育盖玻片上顶。放置玻片在湿盒填充50毫升50％formamide/2x SSC，孵育过夜，在37°C。 </li></ol> 5，杂交后的洗涤和抗体介导的检测探头<ol><li>填6孔板用2×SSC，和预暖至42℃。隔夜培养后加入盖玻片，孵育在42℃下5分钟。 </li><li>除去2×SSC，和孵育盖玻片，用50％formamide/2x SSC中10分钟，在42℃下在重复共3次（共洗30分钟）。 </li&gt; <li>除去50％formamide/2x SSC，并用Tris-生理盐水吐温（TST）为2×5分钟，在室温下洗幻灯片。 </li><li>点50微升的Tris-盐-BSA（TSBSA）每盖玻片上新的载玻片，并孵育盖玻片倒置时30分钟，在室温下，在TST-加湿暗室阻塞。 </li><li>转让盖玻片回6孔板，洗2×5分钟，TST。 </li><li>点50微升羊 - 抗-DIG抗体（以TSBSA 1:500）的每盖玻片上新的载玻片，并在30分钟内于室温下，在TST-加湿暗室第一孵育步骤孵育盖玻片倒置。 </li><li>转让盖玻片回6孔板，洗2×5分钟，TST。 </li><li>点50微升的兔抗羊抗体每盖玻片上新载玻片结合有FITC（在TSBSA 1:250），并孵育盖玻片倒置时30分钟，在室温下，在TST-加湿暗室第二孵育步骤。 </li><li>反式FER盖玻片回6孔板中，并洗涤2×5分钟，用TST。 </li><li>点50微升的山羊抗兔抗体每盖玻片上新载玻片结合有FITC（在TSBSA 1:250），并孵育盖玻片倒置时30分钟，在室温下，在TST-加湿暗室第三孵育步骤。 </li><li>转让盖玻片回6孔板，洗2×5分钟，TST。 </li><li>点50微升小鼠抗生物素抗体（在TSBSA 1:200）每盖玻片上新载玻片，并孵育盖玻片倒置时30分钟，在室温下，在TST-加湿暗室第四孵育步骤。 </li><li>转让盖玻片回6孔板，洗2×5分钟，TST。 </li><li>点50微升的驴抗小鼠抗体的每盖玻片上新载玻片结合有若丹明（在TSBSA 1:250）孵育盖玻片倒置时30分钟，在室温下，在TST-加湿暗CHAMB第五孵育步骤呃。 </li><li>转让盖玻片回6孔板，洗2×5分钟，TST。 </li><li>点50微升的山羊抗马抗体的每盖玻片上新载玻片结合有若丹明（在TSBSA 1:250）孵育盖玻片倒置时30分钟，在室温下，在TST-加湿暗室的潜伏期第六步骤。注意：在山羊抗马抗体将识别驴抗小鼠抗体;提供给研究者时，一个山羊抗驴抗体将正常工作。 </li><li>转让盖玻片回6孔板，洗2×5分钟，TST。用Tris-生理盐水（TS）的额外洗5分钟。 </li><li>通过随后的温育在70％乙醇脱水的细胞为3分钟，90％乙醇中3分钟，100％乙醇中3分钟。从6孔培养板除去盖玻片，并让空气干燥几分钟。 </li><li>发现一个下拉安装介质用DAPI（见试剂）在载玻片上，并加盖玻片上顶倒置。滑动密封用指甲油和通过荧光显微镜评估FISH结果（ 图1）。 </li></ol>

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+action+in+the+X-chromosome+of+the+mouse+(Mus+musculus+L).">Gene action in the X-chromosome of the mouse (Mus musculus L).</a> Nature. 190, 372-373 (1961).</li><li>Barakat, T. S., Gribnau, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=chromosome+inactivation+in+the+cycle+of+life.">chromosome inactivation in the cycle of life.</a> Development. 139, 2085-2089 (2012).</li><li>Augui, 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=Regulation+of+X-chromosome+inactivation+by+the+X-inactivation+centre.">Regulation of X-chromosome inactivation by the X-inactivation centre.</a> Nat Rev Genet. 12, 429-442 (2011).</li><li>Brown, C. J., 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=A+gene+from+the+region+of+the+human+X+inactivation+centre+is+expressed+exclusively+from+the+inactive+X+chromosome.">A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.</a> Nature. 349, 38-44 (1991).</li><li>Brockdorff, N., 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=Conservation+of+position+and+exclusive+expression+of+mouse+Xist+from+the+inactive+X+chromosome.">Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome.</a> Nature. 351, 329-331 (1991).</li><li>Borsani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+gene+expressed+from+the+inactive+X+chromosome.">Characterization of a murine gene expressed from the inactive X chromosome.</a> Nature. 351, 325-329 (1991).</li><li>Barakat, T. S., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+inactivation+and+embryonic+stem+cells.">X chromosome inactivation and embryonic stem cells.</a> Adv Exp Med Biol. 695, 132-154 (2010).</li><li>Chaumeil, J., 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=A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Okamoto, I., 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=Eutherian+mammals+use+diverse+strategies+to+initiate+X-chromosome+inactivation+during+development.">Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development.</a> Nature. 472, 370-374 (2011).</li><li>Gribnau, J., 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=X+chromosome+choice+occurs+independently+of+asynchronous+replication+timing.">X chromosome choice occurs independently of asynchronous replication timing.</a> J Cell Biol. 168, 365-373 (2005).</li><li>Namekawa, S. 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作者什么都没有透露。

结合DNA-RNA鱼可以在技术上具有挑战性，因为适用于DNA FISH条件可能过于苛刻不太稳定的RNA分子。几种方法已经被用于研究DNA和RNA在相同的小区，从而无需采用同时孵育探针检测两者的DNA和RNA。例如，在一个叠加方法中，第一RNA FISH被执行，并且细胞进行成像，并且坐标被采取。随后，相同的载片用于DNA鱼，在此期间，核糖核酸FISH的信号丢失。 DNA FISH后，将细胞再成像，并从二者的RNA和DNA的鱼获得?...

通过荧光的手段原位杂交（FISH）研究细胞和组织有，自20世纪70年代后期1引言，让研究者可以研究基因，染色质组织和基因表达在亚细胞水平。 DNA FISH经常用于细胞遗传学，染色体组型2，癌症诊断3和植入前遗传筛查4，并具有在分子研究5-6中起重要作用，因为它允许在其天然环境中的核酸的检测。通过RNA鱼单细胞表达分析可以检测本地初级转录和非编码RNA的染色体被转录，并提供优势的评估在群体水平上的基因表达的其他技术，例如包括定量RT-PCR，全基因组表达分析或Northern印迹。通过可视化的RNA转录直接从原产他们的网站发起，它已经例如被注意到，基因表达是随机7，和有时是等位基因特异性8。在技术改进甚至允许细胞9-11内单个mRNA分子的检测和定量。 FISH的基本原理包括的小区内的核酸杂交到由高度特异性Watson和Crick碱基配对的装置的核酸探针。该探针可以是直接或间接地检测到，从而导致可在显微镜可视化的一个信号。最初的尝试包括放射性标记的探针，基于安全问题，有限的空间分辨率和探测只有一个目标在12-14时的能力，而这是有缺陷的。的放射性标记，其中包括荧光染料，半抗原和酶的后续发展，已经允许广泛使用FISH技术作为一种常规的分子生物学技术。将FISH探针可以被直接标记与fluorochROMES由荧光分子化学连接到核酸序列15和一体化荧光标记的核苷酸16-19，或者所述探针可以结合的半抗原（包括生物素和地高辛），并通过半抗原缀合的特异性抗体的免疫学检测半抗原后可以间接地可视化的荧光报告分子20-21。后一种方法允许信号放大通过使用用于增强原始信号的荧光标记抗体的若干层，并且使其中被表达在低水平RNA种类的检测。通过结合直接和间接标记技术和各种半抗原，多个目标，可以同时在同一小区内的可视化。 其中一个在鱼的协议中最重要的步骤之一是探测到目标的杂交。虽然在理论上，一个探测器将仅特异性结合其靶标，在实践中，这种特异性并不总是实现，探针可结合到同源的区域，和杂交条件并不总是理想的目标DNA区域或RNA种类。因此，特别重要的是杂交后洗涤的，因为它们可以增加对FISH程序的严格性，并且可以防止FISH探针，这将导致背景噪声较高水平的非特异性结合。如RNA分子是单链，他们可以很容易地杂交到一个FISH探针。与此相反，在双链DNA分子首先需要的变性步骤，在之后的探针可以杂交。这通常是通过加热的试样，这会导致该DNA的变性来实现。然而，这些苛刻的条件下，易碎，单链RNA分子可能会丢失。因此，结合DNA-RNA FISH需要的条件显著优化，并且更具有技术挑战性相比，只有RNA或DNA的分离检测。 在这里，我们PRESEN结合起来，同时DNA-RNA的鱼让我们来研究X染色体失活（XCI）在鉴别雌性小鼠胚胎干细胞22-24的TA详细的协议。 XCI是一个关键的表观遗传机制，为女性胚胎发育25，结果在heterochromatinization，因此沉默，女性26-27个人的两条X染色体中的一个。必不可少的这个过程是在非编码RNA Xist的 28-30，这是由RNF12 22-23和REX1 24蛋白调节。Xist的表达变得期间胚胎发育中或在体外 ES细胞分化的未来失活的X染色体（Xi）的上调，并且可以沿X染色体分散，从而吸引而导致的X染色体31的转录关机染色质重塑酶。这个扩展的Xist RNA可以通过RNA FISH被形象化为在X染色体上的涂层一些，这也被称为Xist的云。由于雌胚胎干细胞可以失去，由于基因组不稳定的X染色体之一，我们和其他人联合聘请DNA-RNA FISH研究XCI，以确保只有稳定的核型细胞在这一重要进程22,32的分析评估-34。如同每分子生物学技术，几种不同的优良协议已经公布35-38。在这里，我们提出我们的方法，从分化的小鼠胚胎干细胞，细胞固定，FISH探针由尼克 - 翻译，前处理固定细胞的标记的诱导开始允许通透和随后的探头摄取，探头到杂交目标，最后检测探头通过荧光标记的抗体。在本文提出的协议允许的忠实检测的Xist RNA和一个为期两天内的X染色体，而这种技术的基础知识可能可以适应加时赛她的系统和研究领域。

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">female mouse embryonic stem cells</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">will be made available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse embryonic fibroblasts</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>can be derived from E13.5 embryos, or via commercial sources e.g Millipore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell culture medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>DMEM, 15% foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 0.1 mM &#223;-mercaptoethanol, and 1000 U ml-1 LIF. Filter sterilize and store at 4&#186;C for maximal 2 weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">LIF</td>
			<td style="width:124px;">Chemicon</td>
			<td style="width:119px;">ESG1107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">DMEM</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">11960085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Fetal Calf Serum&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Penicillin&#8211;streptomycin (100&#215;)</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Non-essential aminoacids&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">&#223;-mercaptoethanol&#160;</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell differentiation medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>IMDM-Glutamax, 15% heat inactivated foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 37.8 &#956;l/l monothioglycerol and 50 &#956;g/ml ascorbic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">IMDM-Glutamax</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">31980-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Ascorbic acid (50 mg/ml)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">monothioglycerol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">T25 cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">690160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">6-well cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">cell culture grade PBS</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Trypsin-EDTA 0.25% (v/v) trypsin/0.2% (w/v) EDTA in PBS&#160;</td>
			<td style="width:124px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">falcon tubes</td>
			<td style="width:124px;">Falcon</td>
			<td align="right" style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">24x24 mm coverslips</td>
			<td style="width:124px;">Menzle</td>
			<td align="right" style="width:119px;">780882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">gelatin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>prepare a 0.2% gelatin/cell culture PBS solution, and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">paraformaldehyde</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>prepare 4% PFA/PBS solutions, by dissolving PFA in PBS. Stir at 70 &#7484;C until completely dissolved and cool down to room temperature. Aliquots can be stored at -20&#7484;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">absolute 100%&#160; ethanol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">32221-2.5L</td>
			<td>use absolute 100% ethanol and RNAse free water to make 70% and 90% ethanol solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">RNAse free water</td>
			<td style="width:124px;">Baxter</td>
			<td>TKF7114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">sterile filter tips</td>
			<td style="width:124px;">Rainin Bio Clean</td>
			<td style="width:119px;">GP-L10F, GP-L200F, RT-1000F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">digoxigenin-labeling kit (DIG-Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745816910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">biotin-labeling kit (Biotin Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745824910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Sephadex G50</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2 ml syringe</td>
			<td style="width:124px;">BD Plastipak</td>
			<td align="right" style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">sterilized cotton</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf tubes</td>
			<td style="width:124px;">Eppendorf</td>
			<td align="right" style="width:119px;">30120086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">yeast tRNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15401-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Salmon Sperm DNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15632-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse cot-1 DNA&#160; 1 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf bench top microcentrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5417R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Eppendorf Refrigerated Centrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5810R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M NaAc, pH 5.6</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">50+ hybridization solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>50% formamide, 2x SSC, 50 mM phosphate buffer pH 7, 10% dextran sulfate pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">formamide</td>
			<td style="width:124px;">Acros organics</td>
			<td align="right" style="width:119px;">3272350000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">dextran sulfate</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">31403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">pepsin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6887</td>
			<td>prepare a 0.2% solution in RNAse free water, and add 84 &#181;l 37% HCL per 100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">HCL, 37% solution&#160;</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">84422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">object glasses</td>
			<td style="width:124px;">Starfrost</td>
			<td style="width:119px;">8037/1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">20x SSC</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">AM9763</td>
			<td>dilute to 2x SSC in RNAse free water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">10 mM Phosphate buffer, pH 7</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>add 57.7 ml of 1M Na2HPO4 and 42.3 ml 1M NaH2PO4; use DEPC treated water and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">denaturation buffer</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>70% Formamide/2xSSC/10mM phosphate buffer pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tween-20</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M Tris pH 7.5 solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tris-Saline-Tween washing solution (TST)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>100 ml 10x Saline, 50 ml 2M Tris, 500 &#181;l Tween-20, add RNAse free water till 1 liter&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">10x Saline solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>dissolve 85 g NaCl in 1 liter RNAse free H2O, and autoclave</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline-BSA (TSBSA)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>500 &#956;l 10x Saline, 250 &#956;l 2 M Tris, 1 ml 100x BSA, 3,25 ml RNAse free H2O</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">BSA, purified, 100x&#160;</td>
			<td style="width:124px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">sheep-anti-dig antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:500 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit-anti-sheep antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-rabbit antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse-anti-biotin antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:200 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey-anti-mouse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-horse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline washing solution (TS)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>10 ml 10x Saline, 5 ml 2 M Tris, 85 ml RNAse free H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting medium with DAPI</td>
			<td style="width:124px;">Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nail-varnish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">fluorescent miscroscope</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined dna-rna fluorescent in situ hybridization (fish) to study x chromosome inactivation in differentiated female mouse embryonic stem cells

荧光原位杂交（FISH）是一种分子技术，它可以在细胞中的核酸的检测。 DNA FISH通常用于细胞遗传学和癌症的诊断，而且可以检测的基因组中，它通常具有重要的临床意义的像差。核糖核酸FISH技术可被用于检测RNA分子在细胞内，并提供了在基因表达调控的重要见解。在同一小区内结合DNA和RNA的鱼在技术上具有挑战性，因为适用于DNA FISH条件可能过于苛刻脆弱的，单链RNA分子。我们在这里提出了一个易于应用协议，它使结合起来，同时检测的Xist RNA和DNA的由X染色体编码。这个结合DNA-RNA FISH协议可能可以应用于其他系统，其中RNA和DNA需要被检测到。

采用上述协议，用于结合DNA-RNA鱼，我们已经能够以可视化的X染色体失活在鉴别雌性的胚胎干细胞， 图1示出了DNA-RNA FISH实验中，我们发现这两种Xist的 （这是一个有代表性的例子作为一个所谓的Xist RNA云在失活的X染色体上和活性X染色体一个基础转录针尖），和X染色体上，这是作为一个定点信号可见的区域中可见。注意，针尖信号中的一个位于所述的Xist云内，由?...

Watch this Scientific Journal Video about Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells at JoVE.com

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

荧光原位杂交（FISH）可在细胞内其天然环境中的核酸的检测。我们在这里描述了一种协议，用于组合，同时检测RNA和DNA的鱼的装置，它可以被用来研究X染色体失活在小鼠胚胎干细胞。

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

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Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Unit 2

Studying DNA and RNA

SCIENTISTS IN ACTION

27.5K 次观看.  Erasmus MC - University Medical Center. 荧光原位杂交（FISH）可在细胞内其天然环境中的核酸的检测。我们在这里描述了一种协议，用于组合，同时检测RNA和DNA的鱼的装置，它可以被用来研究X染色体失活在小鼠胚胎干细胞。 

视频: Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

科研

生物学

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...