在实验室吨的研究是由国家科学基金会（DBI - 0820610和IOS - 1022102）和纽约州卫生署（NYSTEM C024308）的赠款支持。 CM收到来自西班牙教育和科学部（2007-0937）和基金会拉斐尔 - 皮诺，西班牙部博士后。

<ol>
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没有利益冲突的声明。

这里列出的原位杂交方法允许任何感兴趣的基因的时空表达模式的直接可视化的伟大细胞高分辨率，高灵敏度和特异性。该协议不允许之间的基因表达水平的定量比较。然而，该方法的高灵敏度和分辨率，可以揭示一个组织8，18，这是不与其他方法分析基因表达的最可能的基因表达梯度。 该协议包括许多步骤，它可以使故障排除问题。该协议的最重要的步骤是固定的组织和探头的选择和标签。低渗透组织和辛掺入低探头将产生非常微弱的信号，可能很难检测到上述背景。为每个感兴趣的新基因，它是明智的尝试从diffe派生的几个不同的探头租金地区的成绩单。有时候，可能需要两个或三个探测器，针对不同的小区域的基因结合，以获得一个强大的和特定的杂交信号。此外，杂交条件，如温度和杂交缓冲液组成，，可以优化每个基因或组织，以降低或增加杂交紧缩。蛋白酶处理步骤是一个变量，可以改变，以适应不同植物物种或成绩单非常低丰度的检测协议。列入正反两方面的控制探头将有助于查明问题的协议。 石蜡包埋植物组织切片，但稍作修改， 可以在原位杂交用于整个安装过程优化。虽然广泛用于原位杂交动物研究19日 ，整装将升imited到植物组织中，可以很容易地渗透，如胚胎，根或年轻的分生组织20,21。该协议还可以很容易地修改，同时检测两种截然不同的mRNA或本地化一起蛋白 15,22,23的成绩单。最后，它有可能延长这种方法的应用，小分子RNA在植物中的表达模式的可视化。 miRNA表达模式已经确定使用在玉米和拟南芥8,14本议定书。最近， 在体外转录探头已经取代市售高辛标记的锁核酸（LNA），寡核苷酸探针，提高信号灵敏度18，24。

<table border="1"><tbody><tr><th>产品</th><th>公司</th><th>目录编号</th><th>备注</th></tr><tr><td> Cytoseal 604盎司</td><td>费舍尔</td><td> 23-244-256 </td><td>甲苯基于8310-4 </td></tr><tr><td> Histoclear </td><td>费舍尔</td><td> 50-899-90147 </td><td></td></tr><tr><td>组织路径Paraplast加</td><td>费舍尔</td><td> 23-021-400 </td><td></td></tr><tr><td> N +尼龙膜Hybond - N + </td><td> GE医疗集团</td><td> RPN203B </td><td></td></tr><tr><td>核糖核酸酶进行; 40 U /μL </td><td> Invitrogen公司</td><td> 10777019 </td><td></td></tr><tr><td> RQ1无RNase DNA酶; 1 U /μL </td><td> Promega公司</td><td> M6101 </td><td></td></tr><tr><td> DIG标记的RNA控制</td><td>罗氏公司</td><td> 11585746910 </td><td></td></tr><tr><td>辛RNA的标签混合</td><td>罗氏公司</td><td> 11277073910 </td><td></td></tr><tr><td> SP6 RNA聚合酶千ü </td><td>罗氏公司</td><td> 10810274001 </td><tD&gt; </td></tr><tr><td> T3 RNA聚合酶千ü </td><td>罗氏公司</td><td> 11031163001 </td><td></td></tr><tr><td> T7 RNA聚合酶千ü </td><td>罗氏公司</td><td> 10881767001 </td><td></td></tr><tr><td>核糖核酸酶- A </td><td>罗氏公司</td><td> 109142 </td><td>溶于甘油50％三100MM pH8.0 </td></tr><tr><td>阻断试剂</td><td>罗氏公司</td><td> 1 096 176 </td><td>加热到70 ° C的TBS缓冲液</td></tr><tr><td>反Digoxygenin - AP Fab片段，从羊150 U </td><td>罗氏公司</td><td> 1 093 274 </td><td></td></tr><tr><td> NBT / BCIP显原液</td><td>罗氏公司</td><td> 1 681 451 </td><td></td></tr><tr><td>迷你快速旋转的RNA列</td><td>罗氏公司</td><td> 11814427001 </td><td></td></tr><tr><td>的tRNA 从 E 大肠杆菌 </td><td>罗氏公司</td><td> 109541 </td><td></td></tr><tr><td> TRITON ® X - 100 </td><td>西格玛</td><td> T8787 </td><td></td></tr><tr><td>吐温20</td><td>西格玛</td><td> P9416 </td><td></td></tr><tr><td>去离子甲酰胺</td><td>西格玛</td><td> F9037 </td><td></td></tr><tr><td> 灰色链霉菌蛋白酶类型第十四</td><td>西格玛</td><td> P5147 </td><td> milliQ水和预消化4小时稀释在37 ° C </td></tr><tr><td>三乙醇胺</td><td>西格玛</td><td> 90279 </td><td>稀释水，带来用HCl pH8.0 </td></tr><tr><td>醋酐</td><td>西格玛</td><td> A6404 </td><td></td></tr><tr><td> 明串珠菌属硫酸葡聚糖钠盐。 </td><td>西格玛</td><td> D8906 - 5G </td><td>硫酸葡聚糖应加热至80 ° C至解散。 </td></tr><tr><td>登哈特的解决方案50X浓缩</td><td>西格玛</td><td> D2532 </td><td></td></tr><tr><td>牛血清白蛋白 - ≥98％ </td><td>西格玛</td><td> A7906 </td><td></td></tr><tr><td>曙红Y钠盐</td><td>西格玛</td><td> Ë4382 </td><td>在96-100％的乙醇溶解，直至饱和</td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td>乙醇绝对200的证明</td><td></td><td></td><td></td></tr><tr><td>酚/氯仿</td><td></td><td></td><td></td></tr><tr><td>基地模具</td><td>电子Microsocpy科学</td><td> 62352-15 </td><td></td></tr><tr><td>嵌入戒指</td><td>费舍尔</td><td> 22-038-197 </td><td></td></tr><tr><td> 20毫升一次性闪烁瓶，与帽</td><td> VWR </td><td> 66020-326 </td><td></td></tr><tr><td>探测器上加幻灯片</td><td>费舍尔</td><td> 22-230-900 </td><td></td></tr><tr><td>微盖玻片24x50毫米</td><td> VWR </td><td> 48404-452 </td><td></td></tr><tr><td>滤纸纸3毫米</td><td> VWR </td><td> 89022-360 </td><td></td></tr><tr><td>三玻璃菜和一个STAInless钢滑动架</td><td>电子Microsocpy科学</td><td> 71420-25 </td><td>两片玻璃抛出histoclear治疗的必要，最后一个是醋酸酐处理</td></tr><tr><td> 18个干净的塑料容器</td><td>电子Microsocpy科学</td><td> 71402 </td><td></td></tr><tr><td> 2个或3个大平面密封的盖子的塑料盒</td><td></td><td></td><td>需要的杂交和抗体步骤</td></tr><tr><td>精细刷子</td><td></td><td></td><td>帮助移交蜡色带</td></tr><tr><td>切片机</td><td></td><td></td><td>徕卡</td></tr><tr><td>自清洁的干式真空系统</td><td>韦尔奇</td><td> 2025 </td><td></td></tr><tr><td>滑入温暖</td><td>费舍尔</td><td></td><td></td></tr><tr><td>加热块</td><td></td><td></td><td>需要在60 ° C和85 ° C</td></tr><tr><td>在58-60孵化° C </td><td>费舍尔</td><td></td><td>对于与熔融Paraplast的步骤</td></tr><tr><td> 55 ° C和37 ° C的烤箱或水浴</td><td>费舍尔</td><td></td><td>需要两个由于温度之间的快速变化</td></tr><tr><td>离心（4℃ - 20℃） </td><td></td><td></td><td></td></tr><tr><td>通风柜</td><td></td><td></td><td></td></tr></tbody></table>

in situ hybridization for the precise localization of transcripts in plants

随着基因组学研究在过去十年的进步，植物生物学已经看到大量的研究，提出大型基因表达的定量分析。正在使用的芯片和下一代测序方法调查发育，生理应激反应过程，解剖后生和小RNA的途径，并建立大的基因调控网络1-3。虽然这些技术方便的大型基因组的同时分析，他们通常会提供一个非常有限的时空分辨率，基因表达的变化。这种限制可以通过使用分析方法结合lasermicrodissection或荧光激活细胞分选4-7部分克服。然而，要充分认识的一个基因的生物作用，在细胞决议，其表达的时空格局知识是必不可少的。特别是，当研究发展或环境STI影响木里和突变体的一个基因的表达模式的详细分析变得至关重要。例如，在微妙的关键调控基因的表达水平的定量差异可能导致在特定的细胞类型中表达的损失或收益时，戏剧性的表型。 有几种方法经常用于基因表达模式的详细检查。其一是通过转基因记者线的分析。 ，但是，这种分析可以成为耗时分析多个基因或植物顽抗转型工作。此外，一个独立的验证，以确保转基因表达模式模拟的内源性基因，通常是必需的。免疫组化蛋白定位或mRNA原位杂交中，目前基因表达的细胞和组织内的直接可视化的比较快的替代品。后者具有明显的优势，它可以很容易ü任何感兴趣的基因原位杂交。SED允许一个标记的反义RNA杂交细胞与靶mRNA的检测探头在体外感兴趣的基因的转录获得。 在这里，我们为协议大纲在原位定位基因在植物中的表达，是高度的敏感性和具体。它是使用多聚甲醛固定，石蜡包埋切片，这给组织学的优秀保存，地高辛标记探针免疫检测和碱性磷酸酶比色法反应的可视化进行了优化。该协议已成功地应用于广泛的植物物种组织，可用于分析mRNA的以及小分子RNA 8-14表达。

Watch this Scientific Journal Video about In Situ Hybridization for the Precise Localization of Transcripts in Plants at JoVE.com

In Situ Hybridization for the Precise Localization of Transcripts in Plants

“<em&gt;原位</em&gt;这里描述的杂交协议允许的mRNA和小RNA在细胞水平的高灵敏度和特异性表达的直接本地化。石蜡包埋植物组织切片进行了优化的过程，是适用于广泛的植物和组织，并可以在十天内完成。

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 ...

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Biology

In Situ Hybridization for the Precise Localization of Transcripts in Plants

51.1K 次观看.  Cold Spring Harbor Laboratory. “原位这里描述的杂交协议允许的mRNA和小RNA在细胞水平的高灵敏度和特异性表达的直接本地化。石蜡包埋植物组织切片进行了优化的过程，是适用于广泛的植物和组织，并可以在十天内完成。

视频: In Situ Hybridization for the Precise Localization of Transcripts in Plants

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

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the 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 ...

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 ...

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 ...

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 ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.