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.).

全ての動物-関与手順はノッティンガムの倫理審査委員会の大学に従って行われました。 1.免疫染色のための適切な組織標本を選択します<ol><li>以前25記載されているように、野生型CD1マウス胚および成体の脳組織のパラフィン包埋切片を生成します。方法25の免疫染色のために4％ホルムアルデヒド（FA）または4％パラホルムアルデヒド（PFA）のいずれかで固定した脳組織切片を使用します。 注：パラフィン包埋組織切片の使用は、抗体標識の前に脱脂が必要です。 2を用いた治療ので - DNA変性のためのプロトコルで用いられる4 M塩酸（HCl）、最も抗原回復戦略とは互換性がありません、我々は、タンパク質と5MC酸化誘導体の同時検出のために冷凍のかミクロトームセクションのいずれかを使用することをお勧めしますマーカー。 </li></ol> 2.脱ロウパラフィン包埋組織切片&lt;<html> / P&gt; <ol><li>実行中のクラスII安全キャビネット内で、室温で、キシレンで満たされたコプリンジャー中で10分間2回ずつパラフィン包埋組織切片を洗浄します。 注：これは、不完全なパラフィン除去が一貫性のない染色パターンにつながることができますように新鮮なキシレンを使用することが重要です。 </li><li>脱ワックス後、急速に95、75、そして室温で10分間、50％エタノール中で連続して洗浄す​​ることにより組織切片を再水和します。 </li></ol> 3.固定および冷凍のとミクロトーム切片の透過処理<ol><li> RTで15分間、氷冷4％PFAまたは4％FAのいずれかでそれらを置くことによって再水和した組織切片を修正しました。 </li><li>室温で5分間、PBS中のセクション（リン酸緩衝生理食塩水）を洗浄することにより、過剰な固定液を除去します。 </li><li> RTで30分間PBXが充填されたコプリンジャー（PBS中0.5％トリトンX-100）中に配置することによって、組織切片を透過。 PBT（PBS中0.01％のTween 20）でまもなくセクションを洗浄することにより、過剰なPBXを削除してください。 </li></ol>jove_title &quot;&gt; 4。オキシナイト5MC誘導体の免疫染色<ol><li> DNAの脱プリンをRTで60分間、2 N HCl中で透過処理セクションを配置します。 注：より効率的なDNA変性へのHCl（ 例えば、4 N）の鉛のより高い濃度が、これらはDAPIでオキシ-MCSの同時検出のために互換性がありません。 </li><li>塩酸を中和するために、室温で30分間、10mMトリス - 塩酸（pH8.5）中のセクションを配置します。また、PBS中で5分ごとにセクションを3回洗浄します。室温で5分間PBTのセクションをインキュベートします。 </li><li>慎重に組織切片を静かにスライドを振盪することにより、任意のステップで完全に乾燥させることなく、組織切片を周囲から液体を除去します。セクションに触れずにセクションを取り囲むように、疎水性バリアペンを使用してください。 注：疎水性バリアペンは、組織を染色するために必要な抗体の量を減少させ、複数のセクションがdifferenで染色することができるようにすることができます同じスライド上のトン抗体。 </li><li>湿潤チャンバー中、室温で1時間ブロッキング溶液（PBS中10％のウシ血清アルブミン）100μl中のセクションをインキュベートします。 注：このステップをスキップする修正されたシトシン塩基を染色の効率に影響を及ぼさないであろう。 </li><li>マウスモノクローナル抗5hmC 5,000希釈液と1：湿潤チャンバー内で室温で1時間、ブロッキング溶液中で、ウサギポリクローナル抗5caC一次抗体を1000倍希釈液100μlで組織切片をインキュベートします。必要に応じて別の方法として、4℃で一晩のインキュベーションを行います。 注：一次抗体なしで処理セクションは、染色手順のためのような適切な陰性対照を提供することができます。 5caC、5FCと5hmC 20に富むポスト交尾マウス胚性脳切片を陽性対照として使用することができます12.5日。 </li><li>コップで5分間、PBTで満たされたコプリンジャーに各3回セクションを洗浄することにより過剰な抗体を除去RTで林の瓶。 注：洗浄溶液の体積を増加させるバックグラウンド染色を減少させることができます。 </li><li>過剰PBTを削除して、必要に応じて、PBTは、疎水性の障壁を弱めることができ、界面活性剤が含まれているとして、疎水性のバリアペンで再びセクションを取り囲みます。 </li><li>ヤギ抗ウサギHRP結合抗体の400希釈および1：ブロッキング溶液中のロバ抗マウス555結合抗体の400倍希釈してください。 </li><li>湿潤チャンバー中、室温で1時間、 ステップ4.9からの二次抗体混合物100μl中の組織切片をインキュベートします。 </li><li>室温で5分間ずつPBT 3回満たしコプリンジャーに組織切片を洗浄してください。 </li><li>室温で2分間、チラミドシグナル増幅バッファ内のチラミドの200希釈：1100μlの組織切片を置きます。 </li><li> すぐに、PBTで5分間スライドをそれぞれ3回洗浄することによって過剰チラミド溶液を除去。 </li><li>気をつけyが過剰PBTを削除し、 すぐに （材料リストを参照してください）封入剤のドロップを持つセクションをカバーしています。 </li><li>静かに組織切片上のカバースリップを置き、 すぐにマニキュアでカバーガラスをシール。 </li><li>顕微鏡検査の前に、いくつかの時間のために4℃で組織切片を保管してください。 （ - 40X倍率10）405、488および/または555 nmで蛍光顕微鏡下で調べます。 </li></ol>

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The authors declare no conflict of interest.

いくつかの組織で5MC酸化誘導体、5FCと5caCの報告低量は、標準的な免疫化学プロトコルのために重大な制限を提示するであろうが、ペルオキシダーゼ結合二次抗体の取り込みは、固定組織や細胞におけるこれらのシトシン修飾の検出を可能にした（ 図2） 。しかし、チラミド溶液と最適なインキュベーション時間は、信号強度とチラミドベースの信号増幅の期間の間に直線関係が詳細については、観察されたチラミドシグナル増幅キットの各個々のバッチについて実験的に最適化する必要がありアルメイダらを参照してください。2012年26 。さらに、信号/背景比が著しく過剰な抗体の効率的な除去を可能にするためにコプリンジャー中での洗浄を実施することによって向上させることができます。チラミド溶液とのインキュベーション後、直ちにDにPBT溶液中で洗浄することにより反応を停止することが重要ですecreaseバックグラウンド染色。セクションでは、手順の間の任意の時点で乾燥できるようにするために重要ではありません。 DNAの脱プリン化の効率は、37℃で脱プリン反応を行うことにより改善することができます。それは、二本鎖DNAと排他的に相互作用する4 N HClを使用しての代わりに2ながらNは、DAPIで共染色することを認めていないDNAの脱プリン化のために4 N HClを用いて5MCの修飾された形態の染色を強化します。 この技法は、検出可能な、それは5MCまたはその酸化誘導体の絶対的なレベルを評価するために使用することができないシトシン塩基の修飾された形態の強固な半定量的評価を提供することができません。したがって、我々は他の補完の使用をお勧めしますが、量的には16、19 -24に近づきます。哺乳類のゲノム中の5MC酸化誘導体の発見以来、いくつかのアプローチは、それらの生物学的役割16、19-24を研究するために開発されてきました。これらapproaが、CHESは5MC酸化誘導体の絶対レベルを決定する上で貴重なことができ、彼らは彼らの空間分布20、26に関する情報を提供していません。我々は成功し、異なる細胞型で5MC酸化誘導体の空間分布及び局在をマッピングするために、ここで説明した方法を使用していました現像及び成人の脳20のその空間分布20を明らかにすることにより、この技術は、生物学的影響および5MCのこれらの修飾された誘導体は、細胞の分化、開発および疾患を含む検出することができ、様々な生物学的状況における5MCの酸化誘導体の運命を理解するために重要であることができます。また、ここで説明する方法は、tyramiと異なるインキュベーション時間で（染色の強度に比例する）、ペルオキシダーゼ反応の動態を評価することにより、異なる組織における5MCの酸化誘導体の半定量的な評価を与えることができますデ26。

DNA中のシトシン塩基（5MC）のメチル化は、1サイレンシング転写に関連した脊椎動物のゲノム中に見出される主要なエピジェネティックなマークを表します。 5MC導入及び維持DNAメチルトランスフェラーゼ2-5によって、ゲノムインプリンティング、X染色体不活性化、細胞の分化および発達3,6を含む生物学的プロセスの数に重要な役割を果たすことが示されているされている。従って、の破壊を5MCゲノムパターンは疾病7、8月11日の番号に関連付けられています。開発と疾患における5MCの役割を理解する上での進歩にもかかわらず、まだ、このマークを開発し、成体組織では削除されているかほとんどわかっていないされています。 15、14、13、DNA脱メチル化の潜在的なメカニズムは、最近12アクティブおよびパッシブ脱メチル化機構を含む提案されているいくつかの。十から十一の転座enzymによって媒介5MC逐次酸化の製品の発見5- hydroxylmethylcytosineとしてエス（tet1 / 2/3）（5hmC）、真核生物のDNA 16、17、18、19、5-formylcytosine（5FC）、および5-carboxylcytosine（5caC）は、それらの中間体として機能することができるかどうかを推測を促し自分の権利13におけるDNA脱メチル化のプロセスや行為などの安定したエピジェネティックなマーク。塩基除去修復の成分は、チミンDNAグリコシラーゼ（TDG）は、DNA 19から5FCと5caCの両方に結合し、除去することができることを実証しながら、20はアクティブなDNA脱メチル化における修飾5MC誘導体の役割を示唆しています。 5FC / 5caCが転写調節29におけるこれらのマークの潜在的な関与にRNA IIの処理能力の点の速度を調節することができることを示す最近の証拠。 5MCの酸化形態のこの潜在的な生物学的な重要性を、生化学的及び抗体ベースの技術の範囲は、それらのゲノム分布とグローバルコンテンツ16、19-24を研究するために使用されてきました。 トンのほとんどを考えます彼脊椎動物の器官は、異なる細胞型で構成され、修正されたシトシン塩基の分布は、組織及び細胞型特異16-18、20、23、25〜27であることが、種々の組織における酸化5MC誘導体の空間分布を決定することは重要な実験になりますそれらの生物学的機能を発表するために必要なタスク。生化学的および抗体ベースのアプローチのほとんどは、異なる組織および細胞型における5MCの修飾された形態の分布に関するあらゆる空間情報を提供していません。これとは対照的に、免疫化学ベースの技術は5MC 28とその酸化誘導体20の空間分布および核局在化を評価するための迅速なツールを提供することができます。それを言われて、マウスゲノム18における5FC（各10 6シトシン20）と5caC（各10 6シトシン3）の報告が非常に低い存在量は、標準的な免疫化学のための重要な課題です。 ここに、我々は、堅牢提供し、高感度免疫化学的方法および哺乳動物の脳組織中のシトシンの酸化型の迅速な検出を説明します。信号増幅ステップと結合されたペルオキシダーゼ結合二次抗体を組み込むことによって、この方法は、5FCと5caCの非常に少量を検出する課題を回避します。また、この技術は、効果的にこれらのエピジェネティックマークの生物学的機能を解明する他のアプローチを補完し、系統特異的マーカーとシトシンの修飾形態を同時検出するために用いることができます。

<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.

脳組織切片における5hmCの分布を決定するために、我々は、具体的には、このマークが相互作用するが市販の抗5hmC抗体を用いて、有糸分裂後ニューロン、NeuNのためのマーカーで、このエピジェネティック修飾の同時検出を行っていない変更された他の形態でシトシン20、25。成人の脳における5hmCと5caC分布の免疫組織化学的分析は、著名な5hmC染色がのNeuN陽性細胞との共局在のに対し、NeuNの陰性のグリア細胞は、ゲノム5hmC（ 図1）20のより低いレベルを有することが明らかになりました。 我々は最近のTet-依存5MC酸化が神経幹細胞（NSC）20の系統の仕様の間に動作可能であることを示しました。 NSCは、5FCと5caCに免疫化学的に検出不可能であるにもかかわらずニューロンのAに向けてのNSC分化の初期段階で顕著な免疫染色​​を示します<html> NDグリア系統。これらの両方のマークは、一過性の早期のニューロンとグリア分化20のマーカーの出現と同時に蓄積します。我々はグリア分化誘導の3日目にNSCの固定の文化にグリアマーカーGFAPと、このマークの共染色を行ったNSCを分化に5caCの分布を決定すること。 NSC又は成熟アストロサイトとは異なり（データは示さず）、我々は、これらの培養物においてGFAPを発現する細胞（ 図2）20の比較的大きな割合で強い5caCシグナルを観察しました。 <img alt="図1" src="/files/ftp_upload/54416/54416fig1.jpg" /> DAPI（青）で対比成人脳組織内のNeuN（赤）と5hmC（緑）の図1.共同免疫染色。個々のチャネルおよびマージされた図が示されています。スケールバーは25μmです。「ブランク&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/54416/54416fig2.jpg" /> グリア分化の3日目でのNSCの培養におけるGFAPと5caCの図2.共免疫染色。個々のチャネルおよびマージされた図が示されています。スケールバーは20μmである。 <a href="https://www.jove.com/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</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.

高感度免疫組織化学を使用してシトシンの修飾された形態の検出

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 ビュー.  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.

ビデオ: 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 変更染色: 共焦点画像の数値解析

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

遺伝学

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.

のBalb / Cの脳組織の高感度5 hydroxymethylcytosine検出

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

神経科学

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-メチルシトシンと開発の 5-ヒドロキシメチルシトシンの免疫組織化学的検出と後マウス網膜

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 ...

発生生物学

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.

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

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

ユビキチン化、SUMO化、およびADPリボシル化DNA-タンパク質架橋の測定

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.

著者スポットライト:DNAタンパク質架橋とその翻訳後修飾の定量的検出  

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-タンパク質架橋とその翻訳後修飾の定量的検出

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

生化学

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.

ゲノムDNAから5  - ヒドロキシの選択的捕獲

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.

マウスの神経幹細胞と脳における5-ヒドロキシメチルシトシンの検出

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は、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

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

高感度免疫組織化学を使用してシトシンの修飾された形態の検出

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DNA 変更染色: 共焦点画像の数値解析

のBalb / Cの脳組織の高感度5 hydroxymethylcytosine検出

5-メチルシトシンと開発の 5-ヒドロキシメチルシトシンの免疫組織化学的検出と後マウス網膜

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

DNA-タンパク質架橋とその翻訳後修飾の定量的検出

DNA-タンパク質架橋とその翻訳後修飾の定量的検出

DNA-タンパク質架橋とその翻訳後修飾の定量的検出

ゲノムDNAから5 - ヒドロキシの選択的捕獲

マウスの神経幹細胞と脳における5-ヒドロキシメチルシトシンの検出

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、