We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1.探针制备<ol><li>获得在Xist的多个独特的寡核苷酸（20-30核苷酸长度的寡核苷酸，63-65ºC熔化温度， 表1）与5&#39;-氨基修饰和悬浮在水中。 </li><li>具有5&#39;-氨基修饰（总4.5微克），并与胺反应性荧光染料按照制造商的指示标记池等摩尔量的寡核苷酸。 <ol><li>溶解在最后5微升不含核酸酶的水将合并的寡核苷酸并加入3微升的1M碳酸氢钠的寡核苷酸溶液中。 </li><li>添加2微升DMSO中的胺 - 反应性染料和涡流小瓶简要介绍。 </li><li>混合使用在DMSO中的活性染料的寡核苷酸溶液中，在室温下孵育混合物1小时，在黑暗中。 </li></ol></li><li>净化的荧光标记的寡核苷酸探针通过G-25柱，然后用乙醇沉淀。 <ol><li&gt;在750×g离心1分钟，离心G-25柱以除去过量的贮存缓冲液。 </li><li>添加40微升不含核酸酶的水，以在步骤1.2.3中产生的标记混合物中并应用到G-25柱。离心，在750×g离心2分钟。 </li><li>添加50μl的无核酸酶水，10微升3M醋酸钠（pH5.2）和250微升乙醇的纯化的探针从步骤1.3.2。商店在21,000×g离心15分钟，-80℃下30分钟，并离心在4℃下。 </li><li>用1ml的70％乙醇洗一次沉淀的寡核苷酸。 </li><li>重悬在50μl无核酸酶水或TE中（10mM的Tris-HCl，pH为7.5; 1mM EDTA）中为大约10微米的最终浓度。 </li></ol></li><li>稀释的荧光标记的寡核苷酸探针以250nm的带杂交缓冲液的终浓度（10％甲酰胺; 2×SSC [300mM NaCl的30 mM柠檬酸钠]; 2毫克/毫升牛血清白蛋白; 10％硫酸葡聚糖）。 注：有了这个快速FISH协议，寡核苷酸P在Xist的长袍，设计并在更高的浓度比使用寡核苷酸探针，以缩短培养时间为杂交正常FISH协议使用。 </li></ol> 2.将准备注：幻灯片可使用小鼠胚胎干（ES）或胚状体（EB）来制备用于免疫的FISH通过直接在多聚赖氨酸处理过的载玻片要么生长它们或使用细胞离心涂片与细胞悬液分化细胞20,21。这里，示出了滑动制备由细胞离心涂片。 <ol><li>吸在6孔培养皿的培养基。用PBS（室温），并吸彻底清洗。 </li><li>加入0.5 ml胰蛋白酶到6孔培养皿，孵育在37℃下5-10分钟。 </li><li>加入5毫升培养基，并轻轻吹打上下数次分散细胞。 </li><li>在200×g离心转移细胞到一个15毫升的管中，离心5分钟，在室温下进行。 </li><li>丢弃培养基的d轻轻悬浮细胞在2毫升PBS。 </li><li>计数使用血球细胞，并稀释细胞悬浮液，在4×10 5个细胞/ ml用PBS中。 </li><li>组装幻灯片，过滤器卡和剪辑室，并把它们变成了离心涂片转子适当的插槽。添加制备步骤2.6到在细胞离心涂片器的组装单元中的每个腔室各样品的250微升。离心以1500rpm在室温下10分钟。 </li><li>填在冰上3科普林罐用40ml各冰冷PBS，CSK缓冲液（10mM PIPES，pH值6.8; 100mM NaCl的300毫蔗糖; 3毫的MgCl 2）中，并CSKT缓冲液（CSK缓冲液含0.5％的Triton X-100），分别为。 </li><li>后的细胞离心涂片完成时，滑动迅速转移到冰冷的PBS并孵育5分钟。 </li><li>转移滑入冰冷CSK缓冲液孵育1分钟。 </li><li>转移滑入冰冷CSKT缓冲孵育5分钟。 </li><li>传送滑回冰冷CSK BUFFER孵育1分钟。 </li><li>转移滑入4％多聚甲醛的PBS中孵育10分钟，在室温下进行。 </li></ol> 3.免疫<ol><li>将载玻片在科普林缸用PBST（1×PBS中具有0.1％Tween-20）为几秒钟。 </li><li>将幻灯片的倾斜使剩余的缓冲区流下来的幻灯片。小心擦拭过量液体离开滑动，并将其放置在与水的空吸管尖框。覆盖所述滑动用40微升封闭溶液（1×PBS，1％BSA，0.1％吐温-20），轻轻地放在一个盖玻片，并孵育在室温下10分钟。 </li><li>除去盖玻片，除去封闭溶液，并加入40微升稀释到浓度为1的组蛋白H3K27me3初级抗体：在阻断溶液500。放置盖玻片背面上的滑动，并孵育在室温下30分钟。 注：在封闭液核糖核酸酶抑制剂可能有助于维持RNA的RNA这一FIS当使用多克隆抗体ħ协议。 </li><li>洗科普林罐子装满PBST滑动2次，每次5分钟，轻轻晃动边洗。 </li><li>将幻灯片的倾斜使剩余的缓冲区流下来的幻灯片。仔细擦拭多余的液体，然后将滑入提示框。覆盖与40微升第二抗体（1：500）的稀释于封闭溶液中，放置盖玻片，并孵育在黑暗中15分钟。用于以下步骤中，保持在黑暗中的幻灯片在任何时候。 </li><li>在科普林罐子用PBST如在步骤3.4洗涤2次。 </li></ol> 4. RNA FISH与寡核苷酸探针<ol><li>预温热的水的空吸移管尖箱在底部，以防止从在37℃下，在步骤4.3干燥的RNA原位杂交的幻灯片。 </li><li>将幻灯片的倾斜使剩余的缓冲区流下来的幻灯片。小心地从3.6步擦去多余的液体关闭幻灯片，并将幻灯片与笏吸管，提示框呃在底部。加入500微升的FISH洗涤缓冲液（在2×SSC 10％甲酰胺）上滑动，并孵育5分钟，在室温下进行。 </li><li>除去FISH洗涤溶液中，添加8微升的寡核苷酸探针上的滑动，放置盖玻片，并且滑动与水在底部转移到预热的吸管尖盒;孵育在37℃进行1-2小时。 </li><li>放置在水浴中3预热科普林罐，在37℃用FISH洗涤缓冲液，用DAPI FISH洗涤缓冲液，和2×SSC。 </li><li>后1-2小​​时孵育的RNA原位杂交，将滑动到预温热的FISH洗涤缓冲液。 </li><li>孵育滑动几分钟直到盖玻片滑落的幻灯片。 </li><li>将滑动放回洗涤缓冲液，并继续孵育5分钟。 </li><li>幻灯片转移到另一科普林罐预热FISH洗涤缓冲液，用0.5毫微克/毫升的DAPI孵育5分钟。 </li><li>幻灯片转移到另一个科普林罐预热2X SSC孵育5英里ñ。 </li><li>将幻灯片的倾斜使剩余的缓冲区流下来的幻灯片。仔细擦拭多余的液体，然后将重新陷入干燥的提示框。加入4微升抗褪色与DAPI的幻灯片上，并放置盖玻片。 </li><li>密封指甲油盖玻片，并在显微镜下观察。滑动可以储存于4℃在黑暗中为几个月。 </li></ol>

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No conflicts of interest declared.

在本文中，我们提出了一个快速的免疫FISH协议，只需不到5小时即可完成幻灯片的准备，Xist的RNA FISH，和H3K27me3免疫。在与Xist的RNA检测，这通常需要过夜孵育的RNA原位杂交的一般性免疫FISH方法相比，该协议不仅显著降低所花费的时间，而且，提高了使用的寡核苷酸探针的免疫-FISH的灵敏度。 因为Xist的期间的X染色体失活高度转录和其转录被发现强烈聚积在希在顺式 ，很...

哺乳动物的X染色体失活（XCI）是一种策略，以补偿在XX和XY，其中两个X染色体在女性中的一个是转录失活的1之间的X连锁的基因剂量的不平衡。 X染色体失活的长非编码RNA（lncRNA）的研究有很大的模型系统。的X染色体失活是由多个lncRNAs调节，并且已被广泛研究，在过去的几十年中，以揭开lncRNAs，转录，染色质结构和核组织2，3之间的串扰的机制。 位于X染色体的X染色体失活中心（XIC）是一个复杂的遗传基因座由多个基因产生的非编码RNA 4。的X不活动的特定转录物（Xist的）lncRNA在真哺乳亚纲哺乳动物是一个这样的lncRNA起着在X-染色体失活-5,6-了至关重要的作用。 Xist的成绩单环绕夫的位置TURE习近平启动X染色体失活，而当使用RNA FISH可视化显示为云;此形成被称为“Xist的云”7。由于Xist的RNA与各种染色质修饰酶相互作用，共同定位的Xist的云与沉默染色质和镇压转录不同的表观遗传修饰的是在X染色体失活8观察。例如，Xist的RNA相互作用的Polycomb镇压复杂2（PRC2）负责H3K27me3和诱导镇压染色质状态9。在Xist的云兮其共定位与密集的H3K27me3修饰的发生表示曦10,11的兼异的风景线。 细胞遗传学技术，如DNA / RNA FISH用放射性标记探针12最近的和先进的技术，具有增强的灵敏度，已经走过了漫长的道路，从传统的方法荧光成像使用多个寡核苷酸探针13,14。的DNA / RNA的FISH加上免疫已被常规地用作细胞学工具理解时空核组织的RNA定位，染色质结构和修改。最标准的探针制备用于RNA FISH涉及使用质粒或细菌人工染色体（BAC）克隆并随后将其标识或者与切口平移或随机引物15的。然而，近70％的基因在小鼠和基因在人类中的40％的显示的正义和反义转录物16的重叠，因此需要一个链特异的FISH方法以区分义和反义转录物， 在体外转录的RNA探针（核糖探针）通常用于链特异的RNA原位杂交17,18;然而，这涉及到一种制备质粒克隆或PCR产物用T7，SP6中，或T3启动子和合成的核糖核酸探针。此外，核糖探针从GENOM派生IC DNA或cDNA常含有非特定区域和重复的元素，这导致高的背景噪声。另一个问题是，核糖核酸探针，它们是几百个核苷酸的长度，并包含多个荧光团，不能有效地渗透到细胞核中。为了规避这一点，标有在末端的单个荧光多个较短的寡核苷酸探针已经开发出具有良好的灵敏度，均匀的信号强度，并且易于纯化和处理14。此外，DNA寡核苷酸通常比核糖核酸更稳定。我们应用使用Xist的RNA FISH 19寡核苷酸与免疫，了解西安的X染色体失活的过程中诱导Xist的lncRNA的后生动态类似的策略。这个协议描述的创建的寡核苷酸探针和细胞的适当的准备，以及利用免疫荧光和RNA原位杂交的。 Xist的RNA FISH使用多oligonucleotides是在长期成本效益的方法，如果Xist的RNA FISH在自己的实验室常规进行。该技术可用于识别lncRNAs在细胞中同时映射其共定位与表观遗传修饰或因素。该协议的一个主要优点是可以轻松地修改它以满足自己的研究兴趣的能力。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

原位杂交（FISH）用免疫荧光（免疫FISH）结合的RNA的荧光创建可以在单细胞水平被用于检测RNA的定位与同步洞察的空间动态成 ​​蛋白质，表观遗传修饰和其他细节的定位技术这可以通过免疫荧光被突出显示。 X染色体失活是一个范例长非编码RNA（lncRNA）介导的基因沉默。的X不活动的特定转录物（Xist的）lncRNA积累（称为Xist的云）上的两个X染色体在哺乳动物雌性之一是启动的X染色体失活的一个关键步骤。 Xist的核糖核酸直接或间接地与各种染色质修饰酶相互作用并引入不同的后生景观的失活的X染色体（十一）。在西安一个已知的后生特点是组蛋白H3三甲基赖氨酸27（H3K27me3）修改。在这里，我们描述了一个简单而快速的免疫FISH协议利用RNA FISH配上H3K27me3的免疫多个寡核苷酸探针检查Xist的RNA的定位及相关表观遗传修饰检测Xist的RNA。使用寡核苷酸探针的结果在较短的培养时间和更灵敏的检测Xist的核糖核酸的相比在体外转录的RNA探针（核糖探针）。这个协议提供了一个有力的工具用于理解lncRNAs的动力学和其相关联的表观遗传修饰，染色质结构，核组织和转录调控。

快速免疫FISH代表性图像示于图1A。在鉴别女性细胞中检测共定位在熙Xist的RNA云和H3K27me3信号。在第12天时分化，超过90％的EB细胞的有一个Xist的云（ 图1B）。短寡核苷酸探针高效地渗透到了核，导致几乎所有H3K27me3信号共定位与Xist的核糖核酸可视化（ 图1C; 97％，N = 150）。 <img alt="图1" src="/files/ftp_upload/52053/52053f...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

快速荧光<em&gt;原位</em&gt;对于Xist的RNA杂交组合议定书组蛋白修饰免疫荧光X染色体失活

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.8K 次观看.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

视频: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

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

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

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 ...

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 ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...