这项工作是支持的Generalitat加泰罗尼亚（2005 - SGR00437）。作者感谢J.布兰科和维达尔楼，他们的监督和鼓励，并为他的技术援助研究卢卡斯。

一，样品处理和细胞固定<ol><li>留在无菌容器在室温为20分钟，直到液化的精液样本。 </li><li>样品转移到离心管中，并旋转千克5分钟。 </li><li>轻轻取下并丢弃上清液用巴斯德吸管。 </li><li>添加低渗溶液（氯化钾，0.075M）预先加热到37℃，一滴一滴，而上一个旋涡混合，获得了10毫升的最终体积。 </li><li>管置于水浴中在37 ° C 30分钟。 </li><li>在千克离心5分钟。小心丢弃上清液，通过调迁，而不会干扰颗粒。 </li><li> resuspending沉淀后，加入新鲜配制的卡诺固定液（3:1甲醇：冰醋酸）一滴一滴，而上一个旋涡混合获得终体积为8毫升。 </li><li>需要多次重复6分和7个获得白色颗粒。 </li><li>加入新鲜配制的甲醇：冰醋酸（3:1）一滴一滴地调整最终体积取得了良好的细胞扩散（用小颗粒的个案所需的细胞浓度，将几滴而样本足够高的金额精子的复苏，最终体积可以达到1-2毫升）。 ungreased幻灯片拖放到悬浮样品和检查相衬显微镜下，可以通过测试幻灯片。这将让检查获得的细胞分散，将使纠正它，如果它是必要的，进一步的幻灯片，的。 </li><li>为了使扩展，从最低高度40厘米滴两三滴，进入unfrosted幻灯片中心的细胞悬液（先前储存在-20 ° C甲醇脱脂他们）。为了使样品的整个协议中的位置，包含幻灯片用金刚石铅笔对面的精子扩展标记的区域。 </li><li>至少24小时，存放在-20 ° C的幻灯片。 注：除了甲醇：冰醋酸（3:1）一滴一滴地边搅拌是非常重要的一步，以避免形成精子聚集。 </li></ol>二。 Decondensation <ol><li>幻灯片储存在-20 ° C必须解冻继续与协议（在室温下离开）。 </li><li>在连续两个2X生理盐水柠檬酸钠溶液，每次3分钟（2xSSC）coplin罐放置的幻灯片。 </li><li>通过一系列的乙醇洗涤2分钟，在每个coplin JAR传输的幻灯片。开始用70％乙醇，按照90％和100％完成。留在室温下干出来的幻灯片。 </li><li>二硫苏糖醇的解决方案中的幻灯片（1,4 - 二硫苏糖醇5MM，1％Triton X - 100的的2 - 氨基-2 - （羟甲基）-1,3 - 丙二醇50MM）在37 ° C至孵化器decondense染色质。此产品的行为，打破protamines二硫键，线圈在精子细胞核的DNA。这是一个非常重要的一步，因为在精子细胞核的DNA是高度压缩。在二硫苏糖醇（DTT），解决方案的幻灯片的孵化时间必须调整，根据样品的反应本产品。通常情况下，这个时间是8分钟左右，虽然它可以从2至15分钟。 </li><li>随即，转让幻灯片连续两个coplin 2xSSC罐每个coplin 3分钟。 </li><li>继续放置2分钟的幻灯片，通过一系列乙醇洗涤，在每个coplin JAR（70％，90％和100％的乙醇）。让幻灯片在室温下进行干燥。 注意：数码地面电视的过度暴露会导致分散在该过程结束中的鱼类信号，而过短的曝光会导致缺乏某些信号。 </li></ol>三。杂交<ol><li>精子DNA变性在coplin JAR孵化甲酰胺溶液（70％Formamide/2xSSC）在73 ° 5分钟的幻灯片。 </li><li>通过乙醇溶液，每coplin JAR 1分钟（先用70％乙醇，85％和100％完成）转让的幻灯片。离开幻灯片，在室温下进行干燥。 </li><li>直接添加5升的相应准备使用的探针混合到15x15盖玻片（AneuVysion含量多色探针小组：CEP 18/X/Y或LSI 13/21）。 </li><li>因为它是在视频中，小心地放到盖玻片的幻灯片放在一起目标区域和探针混合物。用橡皮泥密封。 </li><li>幻灯片放入预先加热37 °彗星杂交室（HYBrite™），并在37 ° C为6-24小时。 注：鉴于变性的精子样本是强制性的，变性的探针的必要性是由制造商决定 。 在本议定书中所使用的探针混合物准备使用，在这种情况下，不需要探针变性。 根据杂交面积的大小，探针混合物的体积会有所不同。 </li></ol>四。洗杂交后<ol><li>从杂交室取出的幻灯片。 </li><li>取出橡皮泥，小心地拉出盖玻片，轻轻滑动到一侧。 </li><li>为了消除非特异性的杂交信号coplin JAR放置在一个2分钟的幻灯片0.4xSSC/0.3％NP - 40前预热在73 ° C在水浴。 </li><li>传输一个2xSSC/0.1％NP - 40洗解决方案，在室温1分钟的幻灯片。让幻灯片在室温下进行干燥。 </li><li>添加counterstaining产品的目标区域（8个18x18盖玻片L）。最常见的产品中使用的是4&#39;,6 - diamidino - 2 -苯基吲哚（DAPI，Vysis公司）。 </li><li>因为它是在视频中，盖上盖玻片（保持杂交，可以用指甲油密封盖玻片）杂交的地区。 </li><li>商店在黑暗中的杂交幻灯片，在-20 ° C，直到他们的前景分析。在这些条件下的幻灯片，可以存储长达12个月无明显的荧光信号强度丢失。 注：较高的温度或过度的清洗时间可以消除一些信号，而相反的，不会洗出非特异性探针杂交。 counterstaining附加值产品的体积会有所不同，根据杂交面积覆盖的大小。 </li></ol>五，可视化配备一个带通滤波器的DAPI /得克萨斯红/ FITC和单波段水色，FITC和德克萨斯红滤波器三重epifluorescence显微镜下，可以评估的筹备工作。必须遵循的精子细胞核1的正确评价标准的考核标准。 这三种染色体的制备杂交探针与染色体X，Y和18显示的信号。每个正常的精子必须显示一个蓝色信号（对应的18号染色体），和一个绿色的信号（X -轴承精子）或红色的信号（Y -轴承精子）。 在染色体13和21，13号染色体和21号染色体的一个红色信号的绿色信号的探针杂交编制，应加以区别，在每一个正常的精子。

<ol>
	<li>Blanco, J., Egozcue, J., Vidal, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+chromosome+21+disomy+in+human+spermatozoa+as+determined+by+fluorescent+in-situ+hybridization.">Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization.</a> Hum. Reprod. 11, 722-726 (1996).</li></ol>

The authors have nothing to disclose.

本协议描述了如何处理人类获得精子鱼制品的精液样本。使用该协议有可能在雄配子染色体异常分析。精子非整倍体筛查的应用无论是在基础研究方面，在不育男性的生殖意见。虽然在临床诊断中最广泛研究的染色体，X，Y，13，18和21，其他商业探针的染色体和基因位点的广泛可用，也可以在这些实验中使用。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1,4-Dithiothreitol (DTT)</td>
<td>Material</td>
<td>Roche</td>
<td>10197777001</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
<td>Material</td>
<td>Roche</td>
<td>10708976001</td>
<td>&nbsp;</td>
<tr>
<td>Acetic acid</td>
<td>Material</td>
<td>Merck</td>
<td>100.063</td>
<td>&nbsp;</td>
<tr>
<td>AneuVysion&reg; Multicolor DNA Probe Kit<ul style="padding:2px;"><li>20xSaline-Sodium Citrate</li><li>4,6-diamidino-2-phenylindole (DAPI II)</li><li>CEP 18/X/Y DNA probe</li><li>NP-40</li><li>LSI 13/21 DNA probe</li></ul></td>
<td>Material</td>
<td>Vysis Inc.</td>
<td>32-161075 <ul style="padding:2px;"><li>30-805850</li><li>30-804941 </li><li>30-171077</li><li>30-804820</li><li>30-171078</li></ul></td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tube</td>
<td>Material</td>
<td>Nunc</td>
<td>347856</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (glass)</td>
<td>Material</td>
<td>Barloworld Scientific</td>
<td>Hellendahl (ZCT278)</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (plastic)</td>
<td>Material</td>
<td>Deltalab</td>
<td>191087</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (15x15)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600115</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (18x18)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600118</td>
<td>&nbsp;</td>
<tr>
<td>Diamond pencil</td>
<td>Material</td>
<td>Hammacher</td>
<td>335117010</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Material</td>
<td>Merck</td>
<td>100.983</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>Material</td>
<td>Roche</td>
<td>11814320001</td>
<td>&nbsp;</td>
<tr>
<td>Freezer</td>
<td>Tool</td>
<td>Zanussi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gloves</td>
<td>Material</td>
<td>Sanyc</td>
<td>101/3300</td>
<td>&nbsp;</td>
<tr>
<td>HYBrite&trade;</td>
<td>Tool</td>
<td>Vysis Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil</td>
<td>Material</td>
<td>Olympus</td>
<td>35505</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Tool</td>
<td>Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>Material</td>
<td>Merck</td>
<td>106.009</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette</td>
<td>Material</td>
<td>Labsystems</td>
<td>Finnpipette (4500000)</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette replacement tip</td>
<td>Material</td>
<td>Daslab</td>
<td>16-2001</td>
<td>&nbsp;</td>
<tr>
<td>Nail varnish</td>
<td>Material</td>
<td>Quo-cosmetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX60 epifluorescence microscope</td>
<td>Tool</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.</td>
</tr>
<tr>
<td>Pasteur Pipette</td>
<td>Material</td>
<td>Rubilabor</td>
<td>211.0230</td>
<td>&nbsp;</td>
<tr>
<td>Phase contrast microscope</td>
<td>Tool</td>
<td>Nikon Diaphot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic pipette (3ml)</td>
<td>Material</td>
<td>Deltalab</td>
<td>200007</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride (KCl)</td>
<td>Material</td>
<td>Fluka</td>
<td>60128</td>
<td>&nbsp;</td>
<tr>
<td>Rubber cement</td>
<td>Material</td>
<td>Best-test</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4520022</td>
<td>&nbsp;</td>
<tr>
<td>Slide Saver Boxes</td>
<td>Material</td>
<td>Deltalab</td>
<td>19276.1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile container</td>
<td>Material</td>
<td>Deltalab</td>
<td>409726</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Material</td>
<td>PentaFerte</td>
<td>002022300</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>Material</td>
<td>Comark Instruments, Inc</td>
<td>KM12</td>
<td>&nbsp;</td>
<tr>
<td>Tri-distilled water</td>
<td>Material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Material</td>
<td>Sigma</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Material</td>
<td>B/Braun</td>
<td>Aesculap (BD224R)</td>
<td>&nbsp;</td>
<tr>
<td>Vertical laminar flow hood</td>
<td>Tool</td>
<td>Burdinola</td>
<td>OR-ST 1500</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>FB15012</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>Tool</td>
<td>Raypa&reg;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence in situ hybridization (fish) protocol in human sperm

非整倍体是在人类最常见的染色体异常。大多数这些异常的结果在父母gametogenic的过程中减数分裂错误。在人类男性中，这些错误可能会导致精子的生产与数值代表发送这些异常的后代的风险增加的染色体异常。 出于这个原因，荧光原位杂交技术（FISH）技术对精子核，已成为广泛应用于临床诊断的背景下成立一个协议。这种做法提供了一个估计的数值遗传生殖意见征求患者的配子染色体异常的频率。 迄今为止，精子FISH分析中最常见的染色体染色体的X，Ÿ，13，18和21。 此视频文章介绍，分步实施，如何处理和解决人类精液样本，如何decondense和变性的精子染色质，如何继续获得精子的鱼制品，以及如何在显微镜可视化的结果。特别说明的最相关的步骤，以达到最佳效果。

Watch this Scientific Journal Video about Fluorescence in situ hybridization FISH Protocol in Human Sperm at JoVE.com

Fluorescence in situ hybridization FISH Protocol in Human Sperm

此视频文章介绍，分步实施，如何处理精液样本，以实现优质的荧光<em&gt;原位</em&gt;杂交对人类精子。筹备工作得到可用于非整倍体的筛选，在临床诊断中。

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 ...

fluorescence-in-situ-hybridization-fish-protocol-in-human-sperm

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

30.0K 次观看.  Universitat Autónoma de Barcelona. 此视频文章介绍，分步实施，如何处理精液样本，以实现优质的荧光原位杂交对人类精子。筹备工作得到可用于非整倍体的筛选，在临床诊断中。

视频: Fluorescence in situ hybridization FISH Protocol in Human Sperm

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 ...

科研

生物学

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 ...

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 ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

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

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

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

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

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 ...

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

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

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 ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...