这项工作是支持的，CA131967美国国家癌症研究所的资助。

1。 BrdU掺入（末端标记） <ol><li>至约70％汇合在一个150毫米的组织培养皿中的24小时之前加入的BrdU板细胞。 </li><li>用新鲜的完整的媒体，含有20微克/毫升的BrdU（Sigma公司）在适当的时间点，在收获之前更换介质。用BrdU与细胞类型和种类会有所不同，典型的G2期的持续时间在2和5小时（ 图1）之间的介质中培养时细胞的长度。 </li></ol> 2。单层细胞培养染色体的收获<ol><li>删除的文化传媒板块，并收集10毫升，15毫升的离心管中。丢弃剩余的介质。 </li><li>冲洗细胞一次用10ml正矢[或HBSS中（Sigma公司）]。 </li><li>加入5毫升正矢（或HBSS中）中的0.25％胰蛋白酶的板。在室温下孵育，直到细胞脱离板。 </li><li>加入胰蛋白酶处理的细胞悬浮液从步骤2.1的介质中含有10毫升的管。&lt;/ P&gt; <li>离心400 xg离心10分钟以沉淀细胞。吸干上清液，离开0.5毫升介质中加细胞沉淀。 </li><li>用巴斯德吸管移液彻底重悬细胞。 </li><li>加入3滴低渗溶液（0.075 M氯化钾（Sigma公司）温热至37℃下 ），用巴斯德移液管中的细胞悬浮液混合。加入0.5毫升低渗溶液混合，再次用巴斯德吸管。带来的低渗溶液至5毫升的量，并再次混合。在37℃下孵育20-45分钟，这取决于细胞的类型。 </li><li> 400×g离心10分钟，离心。吸干上清液，离开0.5毫升介质中加细胞沉淀。 </li><li>重悬细胞沉淀轻轻轻拂管。在这一点上，渗透细胞肿胀是脆弱的，所以应注意不破坏细胞膜。 </li><li>加3滴混合，轻轻移液用巴斯德pipett卡诺固定液（1:3的冰醋酸酸：甲醇）;E。加入0.5毫升的固定剂和组合，用巴氏吸管轻轻吹打。带上固定液至5毫升的体积，并再次混合，轻轻移液用巴斯德移液管。固定的细胞可以被存储在黑暗中在-20℃下的数个月。 </li><li>离心并吸出，在步骤2.8。 </li><li>细胞沉淀重悬在新鲜固定液;估计的体积的颗粒和附加组件〜10倍的体积的卡诺固定液。 </li><li>添加的细胞悬浮液，逐滴湿，将冰冷的显微镜载玻片上，将载玻片固定在一个〜45°角，将细胞悬浮液，并允许流过的表面的滑动。莱平放在纸巾滑动和洪水的幻灯片，固色剂，在空气中晾干。检查存在的有丝分裂利差用倒置显微镜幻灯片。 </li></ol> 3。核糖核酸酶和乙醇脱水<ol><li>将200μl的10微克/毫升核糖核酸酶A（Sigma）的2倍SSC到每张幻灯片孵育在37 和德克，C 1小时。 </li><li>洗涤滑动3次，用2×SSC，每次3分钟，在室温下的pH值为7.0。 </li><li>通过乙醇（Sigma）的系列（70％，90％和100％），在室温下，每次3分钟脱水幻灯片。 </li><li>空气干燥，在室温下滑动。 </li></ol> 4。 原位杂交探针鸡尾酒BAC加特定染色体着丝粒的计数探针（CEP）或整条染色体涂料的制备<ol><li>对于BAC / CEP同时杂交预杂交，准备两个单独的探头鸡尾酒。我们已经发现，预杂交BAC和CEP探针分别根据不同的严谨的查询结果在以下的背景和更大的信号强度。我们还发现，也可以用来代替BAC克隆个别F粘粒。下面的鸡尾酒配方代表一个单一的幻灯片的体积。如果处理多张幻灯片使用相同的探头简单地增加销量appropr<html> iately。 * BAC DNA探针鸡尾酒： 11.5微升杂交缓冲液（50％甲酰胺（Sigma公司）/ 2个SSC /硫酸葡聚糖（Sigma公司））2μL蒸馏水2 O 2.5微升BAC/Cot1 DNA在DH 2 O（见下面的标签协议）总体积15μL * CEP探头鸡尾酒： 7微升CEP杂交缓冲液（65％Formamide/2x SSC /葡聚糖硫酸酯）2微升DH 2 O 1的微升CEP/Cot1 DNA（与频谱Orange/Cy3/Texas红prelabeled购买）10μl总体积 *在变性和预杂交（5.1）混合BAC探针与CEP探头在3:2的比例25μL/幻灯片和混合探针鸡尾酒。 或</li><li>等分试样20微升/滑动的的全染色体涂料探针（Cy3/Texas红色）成管。继续变性和预杂交步骤。 </li></ol>“&gt; 5。幻灯片和探头变性和原位杂交<ol><li>变性探针鸡尾酒（s）在75℃下 10分钟。预杂交在37℃下30分钟，以允许Cot1的DNA重复序列的杂交，以防止随后的杂交到中期染色体。 </li><li>变性幻灯片科普林氏罐子在70％Formamide/2x SSC，pH值7.0在72°C，持续3分钟。 </li><li>立即脱水幻灯片通过科普林氏罐子中的乙醇系列（70％，90％和100％），在4℃下，每次3分钟。 </li><li>允许的幻灯片空气干燥，在室温下。 </li><li>预暖的幻灯片至45℃，幻灯片上的探针的预杂交（步骤5.1）的最后10分钟的温暖。如果需要预杂交探针鸡尾酒混合，并加入25微升到每张幻灯片，轻轻将每张幻灯片上，并沿边缘用橡皮泥密封盖玻片。在37℃下孵育过夜，在潮湿的腔室，以防止蒸发。 </li></ol><ol><li>洗净滑动科普林氏罐3次，每次3分钟，在38-40℃下在pH值为7.0×SSC，50％formamide/2x。的洗涤液的温度，可能会发生变化的探针之间。如果有高背景杂交可以增加的洗涤液的温度。反之，如果信号微弱，没有任何背景，那么你可以减少这些清洗的温度。 </li><li>洗幻灯片1 PN缓冲的时间，在室温下 3分钟，然后继续的BrdU检测步骤。 </li></ol> 7。的BrdU检测<ol><li>封锁PNM缓冲液在室温下在黑暗中10分钟（200微升/幻灯片）的幻灯片。 </li><li>排水幻灯片，打开边缘，在纸巾上。加入100微升抗BrdU-FITC（50μg/ ml的罗氏或Millipore）在PNM缓冲液30分钟，在37℃下。 </li><li>洗净幻灯片PN缓冲液中，在室温下的3次，每次3分钟。 </li><li>排水过剩的PN缓冲滑动打开边缘纸巾上的时间。加入20μlDAPI /的抗褪色安装的解决方案（Invitrogen公司）。 量的幻灯片用纸巾摁倒在盖玻片给逼出来的气泡和多余的安装解决方案。进行图像分析。 </li></ol> 8。捕捉图像和量化BrdU掺入<ol><li>与奥林巴斯BX61荧光显微镜和CCD相机奥林巴斯100倍的目标，自动过滤器轮和“Cytovision软件（应用影像）拍摄的图像。尿嘧啶被捕捉使用的FITC过滤器;染色体油漆（德克萨斯红，Metasystems或Cytocell）的，BAC的（Cy3标记）和CEP（Vysis频谱橙色）探头被捕获使用一个得克萨斯州红或Cy3的过滤器; DAPI被抓获一个DAPI过滤器（见图2和图3）。 </li><li>个人感兴趣的染色体识别中的BAC / CEP或染色体特异性漆探针（ 图2B和图3B）。利用Cytovision软件，感兴趣的每个染色体是“切出”作为一个整体从中期扩展，一条线绘制通过沿其长度从短臂的染色体长臂（ 图2C和3C）。 Cytovision软件用于沿每个染色体的长度，以量化的像素强度的档案。 Cytovision软件计算的象素所占用的面积，和量化的BrdU或DAPI每个隔离染色体上的信号表示的像素的强度。的那些像素所占据的面积，以获得总的像素数（ 表1和图4），然后乘以每个染色体所表示的平均像素强度。此外，该协议允许最新的复制的染色体区域的可视化。因此，条带状图案的BrdU掺入允许用于检测染色体积极复制区域，并且在复制中的定时的差异之间彩色石印青梅对被视为在此带型（ 图2和图3）的差异。最后，在结合模式的这些差异可以比已知的每个染色体6,7，它允许对同源染色体或染色体臂4之间的定时差的估计值的复制的复制时间地图。例如，两个染色体6的在图3A中显示的差&gt; 2小时的带型时相比，第6号染色体4正常的复制定时。 此外，此处所述的相似的方法已被成功地用于研究的异步复制定时之间的无效和有效的X染色体8,9。确定了使用X染色体涂料的活性和失活的X染色体。画像购入使用QUIPS mFISH软件（Vysis），和X染色体所占据的像素的数目和由荧光拉占用的像素的数目与等量的BrdU，然后计算使用NIH图像（ <a href="http://rsb.info.nih.gov/nihimage" target="_blank">http://rsb.info.nih.gov/nih-image/index.html</a> ）的。 </li></ol> 9。尼克翻译BAC DNA荧光标记（Vysis） <ol><li>下面的探针混合物冷却微型离心管： 70微升（4微克DNA）在卫生署2 O 10微升0.2毫米的dUTP，橙色或绿色 20微升0.1毫米dTTP的 20微升10倍尼克翻译缓冲液 40微升NT的dNTP（减dTTP的） 40微升的缺口转换酶 总体积200μL 孵育@ 16°CO / N。停止在70℃下加热反应10分钟。冰上冷却5分钟。 </li><li>通过添加以下的探针混合物，用乙醇沉淀DNA： 200μL的探测解决方案 48微升3 M醋酸钠 160，微升Cot1DNA（0.25微克/ UL） 1200μL100％的乙醇</li><li>储存在-80℃下为10分钟到O / N。 </li><li>自旋IŇ冷以12,000 xg离心20分钟。倾出乙醇。清洗1次，用70％的乙醇。旋前。倾出乙醇。空气干燥，直至乙醇蒸发。重悬在40μl的蒸馏水2 O的100纳克/微升的BAC DNA的最终浓度。 </li></ol>

<ol>
	<li>Payer, B., 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=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. 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没有利益冲突的声明。

染色体利差的准备是成功的复制时序分析的关键步骤这里描述。列入的秋水仙胺预​​处理步骤之前，低渗处理可能有助于在频率和扩频的有丝分裂细胞。我们通常会公开细胞，以colcemdi的1-3小时收获前，和使用秋水仙碱，终浓度为10微克/毫升。然而，列入秋水仙碱预处理步骤可能会改变G2的长度，因此可能会改变明显凝结染色体的复制时间和状态。 此过程可以应用?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>抗BrdU-FITC </td><td>罗氏微孔</td><td> 11202593001 MAB326F </td><td> 50微克/微升</td></tr><tr><td>尼克翻译工具包</td><td>雅培分子（Vysis） </td><td> 07J00-0001 </td><td></td></tr><tr><td>频谱橙色的dUTP </td><td>雅培分子（Vysis） </td><td> 02N33-050 </td><td></td></tr><tr><td> CEP </td><td>雅培分子（Vysis） </td><td>而异</td><td></td></tr><tr><td> LSI / WCP杂交的缓冲区</td><td>雅培分子（Vysis） </td><td> 06J67-011 </td><td></td></tr><tr><td> CEP杂交缓冲液</td><td>雅培分子（Vysis） </td><td> 07J36-001 </td><td></td></tr><tr><td>染色体排新界南总区</td><td> MetaSystems集团</td><td> D-14NN-050-TR </td><td></td></tr><tr><td>奥林巴斯BX61荧光显微镜</td><td>奥林巴斯</td><td> BX61TRF-1-5 </td><td></td></tr><tr><td>显微镜成像软件系统</td><td>应用成像</td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td>数码相机</td><td>奥林巴斯</td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> 原位杂交食谱 甲酰胺解决方案 70％Formamide/2x SSC 35毫升甲酰胺（Sigma公司） 10毫升10倍SSC 5毫升D 2 H 2 0 pH值至7.0，用HCl（Sigma）的 *重要的是使用甲酰胺已被存储于-20℃下长时间室温下存放会产生甲酸和PH W生病太低。 50％Formamide/2x SSC 25毫升甲酰胺（Sigma）的 10毫升10倍SSC 15 ML DH 2 O pH值至7.0，用HCl（Sigma）的 20×SSC，4 L 702克氯化钠（Sigma公司） 358克柠檬酸钠（Sigma公司） DH 2 O定容 PN缓冲区[0.1中号NaP0 4 0.1％NP_40（Sigma公司）] 请的0.1M溶液每个磷酸钠（过滤器消毒和储存在500毫升的等分试样）。 的0.1M的NaH 2 PO 4，1升 13.8克的NaH 2 PO 4（Sigma公司）： DH 2 O定容 0.1 M的NaH 2 PO 4 1大号 </strONG&gt; 14.2克的NaH 2 PO 4（Sigma公司） DH 2 O量。 PN：0.1 M的pH值调整的Na 2 HPO 4 pH值至8.0 .1 M的NaH 2 PO 4。过滤消毒，加1毫升的NP-40。 PNM50毫升 1.25克非脂肪干奶（Sigma）的 25毫升PN缓冲液（配方以上） 混合15-20分钟，并不断搅拌。附带的2倍，在400×g离心10分钟。使用上清，确保不打扰沉淀牛奶中的蛋白质。 </td></tr></tbody></table>

chromosome replicating timing combined with fluorescent in situ hybridization

哺乳动物DNA复制的启动在染色体上的多个站点在不同的时间在S期，随着时间的复制程序。被认为是一个动态的过程受反应的表观遗传修饰的组织特异性和发展线索，规范的复制时间。但是，调节，当启动染色体上DNA复制的机制仍然知之甚少。同源染色体的复制同步，但有显着的例外情况。例如，在雌性哺乳动物细胞中的两个X染色体中的一个成为后期复制经历一个过程被称为X染色体失活1。除了这种延迟复制时间，估计要2-3小时，一个X染色体上，成为大多数基因转录沉默。此外，一个离散的顺式作用位点，被称为X染色体失活中心，调节此X染色体失活过程中，包括第Ë感应对整个失活的X染色体的复制时间延迟。此外，一些在肿瘤细胞和细胞中的染色体重排暴露于电离辐射，显示复制时间&gt; 3小时，影响了整个染色体2,3显着延迟。我们的实验室最近的工作表明，中断的离散顺式作用常染色体位点在极晚，影响了整个第4号染色体的复制型。额外的染色体工程的研究表明，某些染色体重排，影响了许多不同的染色体造成这种不正常的复制计时表型，这表明所有的哺乳动物染色体包含离散的顺式作用位点，控制适当的个体染色体的复制时间。 在这里，我们提出了一个方法的定量分析结合荧光原位杂交染色体复制时间。这方法允许在同一细胞同源染色体之间的复制时间的直接比较，并被改编6。此外，这种方法可以明确识别的染色体重排，与影响整个染色体的复制时间变化的。这种方法有优势，最近开发的高通量微阵列或不能区分同源基因上的重新排列和重排染色体的测序协议。此外，因为这里描述的方法的计算结果单细胞，它可以检测染色体重排，在一个群体中只有一小部分的细胞中存在的染色体复制定时变化。

人染色体6的复制定时分析的一个例子示出在图2中。细胞中含有ASAR6基因4，位于6q16.1删除，被暴露5小时的BrdU的有丝分裂细胞，收获和加工FISH染色体6油漆探针（Vysis）和BrdU掺入。请注意，有是一个显著差的的BrdU条带图案的2 6的之间，这是与一个延迟复制定时&gt; 2小时中的染色体6的[见4的复制定时绑扎6号染色体模式之一相一致之前删除。此外，有一个显着差异时BrdU掺?...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

染色体复制定时分析的定量方法进行说明。的方法，利用结合BrdU掺入荧光<em在原位</em&gt;，杂交（FISH），以评估哺乳动物染色体的复制定时。这种技术允许在同一细胞重新排列和未重排的染色体直接比较。

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 ...

chromosome-replicating-timing-combined-with-fluorescent-situ

Research

JoVE Journal

Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

13.9K 次观看.  Oregon Health & Science University. 染色体复制定时分析的定量方法进行说明。的方法，利用结合BrdU掺入荧光

视频: Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

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 ...

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 ...

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 ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

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 ...

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 ...

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 ...