We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

所有动物参与的程序是按照英国诺丁汉大学的伦理审查委员会进行。 1.选择用于免疫染色合适的组织准备<ol><li>如先前25描述的生成的野生型CD1小鼠胚胎和成人脑组织的石蜡包埋部分。使用固定为4％甲醛（FA）或4％多聚甲醛（PFA）的免疫染色方法25的脑组织切片。 注:使用的石蜡包埋组织切片之前必须获得抗体标记脱蜡。由于有2个治疗 - 4中的协议，用于DNA变性采用1M盐酸（HCl）的不与大多数抗原修复策略的兼容性，我们推荐使用的联合检测5MC氧化衍生物与蛋白质无论是永冻或切片机切片的标记。 </li></ol> 2.脱蜡石蜡包埋组织切片&lt;<html> / P&gt; <ol><li>在II类运行安全柜，洗石蜡包埋组织切片在科普林罐子装满二甲苯，2次，每次10分钟在室温。 注:为未完成清蜡会导致不一致的染色模式，是用新鲜二甲苯重要。 </li><li>脱蜡后，迅速再水化通过洗涤的组织切片连续在95，75，并在RT每10分钟50％的乙醇。 </li></ol> 3.固定和永冻和切片机切片的透<ol><li>将它们放置在任何冰冷的4％PFA或4％FA在室温为15分钟修复再水化组织切片。 </li><li>通过在RT洗涤在PBS 5分钟的节（磷酸盐缓冲盐水）除去过量的固定剂。 </li><li>通过在科普林缸（在PBS中0.5％的Triton X-100）填充有PBX在R​​T 30分钟将透化的组织切片。在PBT（0.01％吐温20的PBS）洗不久的部分去除多余的PBX。 </li></ol>jove_title"&gt; 4。免疫染色OXI-5MC衍生品<ol><li>放置在2当量盐酸的透截面在RT 60分钟为的DNA脱嘌呤。 注意:虽然高浓度的盐酸（ 例如，4 N），导致更有效的DNA变性，它们不是用于共检测OXI-MCS的用DAPI兼容。 </li><li>放置在10mM的Tris-HCl（pH为8.5）进行30分钟的部分在RT以中和盐酸。可替代地，洗部分三次，每次在PBS中5分钟。孵育在PBT中的章节在RT 5分钟。 </li><li>小心地从周围组织部分区域的液体而不会被轻轻摇晃幻灯片让组织切片完全干燥在任何步骤。使用疏水性障碍笔包围部分不接触部分。 注:疏水屏障笔减少抗体染色的组织可以允许多个部分与differen染色所需的音量同一张幻灯片上牛逼的抗体。 </li><li>孵育在潮湿的腔室中加入100μl阻断溶液（在PBS中的10％牛血清白蛋白）在RT 1小时的部分。 注:跳过此步骤将不会影响染色的修饰的胞嘧啶碱基的效率。 </li><li>孵育在100μl1的组织切片:5000稀释的小鼠单克隆抗5hmC和一个1:在在潮湿室阻挡在RT 1小时溶液兔多克隆抗5caC初级抗体1,000稀释。可替代地，如果需要，过夜，在4℃下进行温育。 注:没有初级抗体处理的部分可以作为适合阴性对照的染色过程。 12.5天后富集5caC，5FC和5hmC 20可以用作阳性对照交配小鼠胚胎的脑切片。 </li><li>通过三次洗涤部分在科普林罐子装满PBT，每次5分钟在警察去除多余的抗体林瓶在室温。 注意:增加洗涤溶液的体积可以减少背景染色。 </li><li>除去过量的PBT和，如果需要的话，与疏水屏障笔再次包围的部分作为PBT的含有洗涤剂，可以削弱疏水屏障。 </li><li>使1:山羊抗兔HRP偶联抗体的1:400稀释并以1:在阻断溶液400稀释的驴抗小鼠555缀合的抗体。 </li><li>从步骤4.9孵育在100μl第二抗体混合物的组织切片在室温在潮湿室中1小时。 </li><li>洗在科普林罐子装满PBT三次，每次5分钟，在RT下的组织切片。 </li><li>放置在100μl1的组织切片:在酪酰胺信号放大缓冲器酪酰胺的1:200稀释，在室温2分钟。 </li><li> 立即，由每个洗涤载玻片3次5分钟在PBT中除去过量的酪酰胺溶液。 </li><li>小心ý除去过量PBT和立即覆盖部分带有一个安装介质的压降（见材料清单）。 </li><li>轻轻地放在组织切片盖玻片，并立即密封指甲油盖玻片。 </li><li>存放在4℃的组织切片用显微镜检验前几个小时。 （ - 40X放大率10）在405，488和/或555纳米的荧光显微镜下检查。 </li></ol>

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The authors declare no conflict of interest.

虽然报告的低丰度5MC氧化衍生物，5FC和5caC在一些组织的将提出显著限制为一个标准免疫化学协议，过氧化物酶缀合的二抗的掺入允许这些胞嘧啶修饰在固定组织和细胞的检测（ 图2） 。然而，最适孵育时间与酪酰胺溶液应当通过实验为每个单独的批次，其中信号强度与酪胺根据信号放大的持续时间之间的线性关系被观察到的，对于细节酪酰胺信号放大试剂盒的优化参阅Almeida 等人。2012 26 。另外，信号/背景比，可以显著通过携带在一个科普林缸中洗涤，以允许有效地除去多余的抗体的提高。在与酪胺液孵化，它立即停止利用水洗PBT解决方案到D的反应是非常重要的ecrease背景染色。关键是不要让部分在操作过程中的任何点干燥。 DNA脱嘌呤的效率可以通过进行在37℃的脱嘌呤反应加以改进。在使用4当量盐酸代替2 N增强了使用4当量盐酸进行DNA脱嘌呤将不允许共染色用DAPI 5MC的修饰形式的染色，因为它与双链DNA完全相互作用。 尽管这种技术可以提供的胞嘧啶碱基，其中可检测的修饰形式健壮半定量评估，它不能被用于评估5MC或其氧化衍生物的绝对水平。因此，我们建议使用其他补充的，但定量方法16，19 -24。因为在哺乳动物基因组5MC氧化衍生物的发现，几种方法已经被开发，以研究它们的生物作用16，19-24。虽然这些approaCHES可以是在确定5MC氧化衍生物的绝对水平有价值的，它们不提供有关的信息的空间分布20，26，我们已成功地使用这里所描述的方法来映射5MC氧化衍生物在不同的细胞类型的空间分布和定位发展中国家和成人大脑20 通过揭示它们的空间分布20中 ，该技术可以是对理解生物影响，并在不同的生物的上下文5MC的氧化衍生物，其中5MC的这些修饰的衍生物可包括细胞分化，发育和疾病进行检测的命运至关重要。此外，我们在这里描述的方法可以通过在含有tyrami不同的孵育时间评估过氧化物酶反应的动力学（其正比于染色强度）得到5MC的在不同组织中的氧化衍生物的半定量评估德26。

在DNA（5MC）胞嘧啶碱基甲基化代表与转录沉默1个相关基因脊椎动物中发现了一个重要的表观遗传标记。 5MC被引入并通过DNA甲基2-5保持，并已被证明在许多生物过程中起重要作用，包括基因组印记，X染色体失活，细胞分化，发育3,6。因此，破坏5MC基因组的图案与许多疾病7，8-11相关联。尽管在理解发育和疾病5MC作用的进展，但仍仍是未知如何标记在发展中国家和成人组织中被删除。 DNA去甲基化的几个潜在的机制近来已经提出包括主动和被动去甲基化机构12，13，14，15。发现5MC连续氧化由一零一一年易位enzym介导的产品ES（TET1 / 2/3），例如5- hydroxylmethylcytosine（5hmC），5- formylcytosine（5FC），和在真核生物DNA 16，17，18，19提示猜测5- carboxylcytosine（5caC）是否可作为中间体在自己的权利13 DNA去甲基化的过程，或者作为稳定表观遗传标记。而碱基切除修复的组件，胸腺嘧啶DNA糖基化酶（TDG）可结合和从DNA的19删除这两个5FC和5caC示范，20表明了改性5MC衍生物在活性DNA去甲基化的作用。最近的证据表明5FC / 5caC可以调节的RNA II延伸能力点率转录调控29这些标记可能参与。由于5MC的氧化形式这个潜在的生物重要性，一系列的生物化学和基于抗体的技术已被用于研究其基因组分布和全球内容16，19-24。 鉴于大多数的t他脊椎动物的器官由不同类型的细胞和的修饰的胞嘧啶碱基的分布是组织和细胞类型特异性16-18，20，23，25-27，在确定在不同组织中的氧化5MC衍生物的空间分布成为一个重要的实验为揭开它们的生物学功能任务必需的。大部分的生物化学和基于抗体的方法并不提供关于5MC的不同组织和细胞类型的修饰形式的分布的任何空间信息。相反，基于免疫化学技术可以提供用于评估5MC 28及其氧化衍生物20的空间分布和核定位一个快速的工具。即表示，所报告的丰富度很低5FC（20中每10 6个胞嘧啶）和5caC（3中每10 6个胞嘧啶）在小鼠基因组18代表用于标准免疫化学一个显著挑战。 这里，我们描述提供了强大的一个高度敏感的免疫化学方法和在哺乳动物脑组织胞嘧啶氧化形式的快速检测。通过将加上信号放大步骤过氧化物酶缀合的第二抗体，这种方法绕过了检测非常低量的5FC和5caC的挑战。此外，这种技术可用于与谱系特异性标记胞嘧啶的共同检测修饰形式，有效地补充在阐明这些表观遗传标记的生物学功能的其他方法。

<table><tbody><tr><td>Coplin jar</td><td>Cole-Parmer</td><td>UY-48585-30</td><td>Glass made</td></tr><tr><td>Xylene</td><td></td><td></td><td>Use only in Class II safety cabinet</td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Fisher Scientific</td><td>12821680</td><td>pH 7.5, filtered before use</td></tr><tr><td>4% paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>P6148</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>4% formaldehyde&amp;nbsp; (FA)</td><td>Sigma Aldrich</td><td>F8775</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>Ethanol, anhydrous denatured, histological grade</td><td>Sigma Aldrich</td><td>64-17-5</td><td>95, 75, 50% in PBS</td></tr><tr><td>0.01% Tween 20 in PBS</td><td>Sigma Aldrich</td><td>P9416</td><td>PBT</td></tr><tr><td>0.5% Triton X-100 in PBS</td><td>Sigma Aldrich</td><td>X100</td><td>PBX</td></tr><tr><td>2 N HCl</td><td>Sigma Aldrich</td><td>71826</td><td>caution extremely toxic</td></tr><tr><td>100 mM Tris-HCl&amp;nbsp;</td><td>Promega</td><td>H5121</td><td>pH adjusted to 8.5</td></tr><tr><td>hydrophobic barrier pen</td><td>Abcam</td><td>ab2601</td><td>for immunohistochemistry</td></tr><tr><td>Slide moisture chamber</td><td>Scientific Device Laboratory</td><td>197-BL</td><td></td></tr><tr><td>anti-5mC mouse antibody</td><td>Diagenode</td><td>C15200081</td><td>monoclonal primary antibody</td></tr><tr><td>Anti-5hmC antibody</td><td>Active Motif</td><td>39791</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Anti-5caC antibody</td><td>Active Motif</td><td>61225</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Peroxidase-conjugated anti-rabbit seconday antibody</td><td>Dako</td><td>K1497</td><td></td></tr><tr><td>555-congjuated goat anti-mouse antibody</td><td>Molecular probes</td><td>A-11005</td><td>&amp;nbsp;secondary antibody</td></tr><tr><td>10% BSA</td><td>Sigma Aldrich</td><td>A9418</td><td>Blocking solution</td></tr><tr><td>22 x 32 mm Glass cover slips</td><td>BDH</td><td>406/0188/24</td><td></td></tr><tr><td>Tyramide Signal Amplification System</td><td>Perkin Elmer</td><td>NEL741001KT</td><td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Colourless nail polish</td><td></td><td></td><td></td></tr><tr><td>chicken polyclonal anti-GFAP polyclonal antibody</td><td>Thermo Scientific</td><td>PA5-18598</td><td>1:400 dilution</td></tr><tr><td>anti-NeuN mouse monoclonal antibody</td><td>Merck milipore</td><td>MAB377B</td><td>1:400 dilution</td></tr></tbody></table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

为了确定5hmC在脑组织切片的分布情况，我们进行了联合检测这表观遗传修饰的与有丝分裂后神经元的NeuN标记，使用专门带有该标志进行交互，而是商业的抗5hmC抗体不与其他形式的修改胞嘧啶20,25。在成人脑5hmC和5caC分布的免疫组织化学分析显示，而突出5hmC染色的NeuN阳性细胞共定位，的NeuN阴性的神经胶质细胞具有基因组5hmC（ 图1）20的下级。 我们最近发现，四环素依赖5MC氧化是神经干细胞（NSCs）20的谱系规范中执行。尽管是在神经干细胞免疫化学检测不到，5FC和5caC表现出对在一个神经元的神经干细胞分化的早期阶段，突出免疫<html>第二胶质谱系。这两个标志瞬时早期神经元和神经胶质分化20标记的外观同时积累。为了确定分化我们在胶质分化诱导后3天对NSCs的固定培养物进行该标记的共染色用胶质标志物GFAP的NSCs 5caC的分布。不像在神经干细胞或成熟的星形胶质细胞（数据未显示），我们在比较大的比例在这些培养物中表达GFAP的细胞（ 图2）20的观察强烈5caC信号。 <img alt="图1" src="/files/ftp_upload/54416/54416fig1.jpg" /> 5hmC在用DAPI（蓝色）复染色成人脑组织（绿色）用的NeuN（红色），个别信道和合并的视图的图1共同免疫染色所示。比例尺是25微米。空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54416/54416fig2.jpg" /> 图5caC的与胶质分化的第3天GFAP在NSC文化2.联合免疫，个别渠 ​​道和合并视图中显示。比例尺是20微米。 <a href="https://www.jove.com/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

detection-modified-forms-cytosine-using-sensitive

Research

JoVE Journal

Biology

7.2K 次观看.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

视频: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

科研

遗传学

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

神经科学

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

发育生物学

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

生物化学

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

生物学

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

胞嘧啶修饰形式使用敏感免疫组化检测

摘要

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此视频中的章节

DNA 修饰的免疫: 共焦图像的计算分析

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

从基因组DNA选择性捕获的5 - 羟甲基

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

胞嘧啶修饰形式使用敏感免疫组化检测

摘要

探索更多视频

此视频中的章节

DNA 修饰的免疫: 共焦图像的计算分析

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

从基因组DNA选择性捕获的5 - 羟甲基

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色