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><tbody><tr><td>oligonucleotides with 5&amp;rsquo;-amino modification</td><td>IDT</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Reactive Dye</td><td>Life Technologies</td><td>A32750</td><td></td></tr><tr><td>MicroSpin G-25 columns</td><td>GE Healthcare</td><td>27-5325-01</td><td></td></tr><tr><td>Cytospin 2</td><td>Shandon</td><td>59900102</td><td></td></tr><tr><td>Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td><td>Roche</td><td>10715859103</td><td></td></tr><tr><td>Histone H3K27me3 antibody</td><td>Active Motif</td><td>61017</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td><td>Life Technologies</td><td>A21424</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36931</td><td></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

13.9K 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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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 ...

生物学

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

利用荧光 原位 杂交探索卵巢细胞中的X染色体畸变

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

遗传学

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

神经科学

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

免疫与感染

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

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

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

Combined 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 ...

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.

快速荧光

摘要

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此视频中的章节

了解早期器官使用简体

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

斑马鱼整装高分辨率双荧光原位杂交

利用荧光原位杂交探索卵巢细胞中的X染色体畸变

荧光钙成像及其后续原位杂交，用于异种性前体特征

结合双荧光

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

全山RNA荧光

结合DNA-RNA荧光

对于荧光DNA的简易方法

快速荧光

摘要

探索更多视频

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了解早期器官使用简体

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

斑马鱼整装高分辨率双荧光原位杂交

利用荧光 原位 杂交探索卵巢细胞中的X染色体畸变

荧光钙成像及其后续原位杂交，用于异种性前体特征

结合双荧光

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

全山RNA荧光

结合DNA-RNA荧光

对于荧光DNA的简易方法

利用荧光原位杂交探索卵巢细胞中的X染色体畸变