Name: combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization
Uploaded: 2013-02-03T14:00:00
Description: Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

我々は、議論とコメントのためのスザンナ·ヒューイット、特に、Skokラボのメンバーに感謝したいと思います。この作品は、健康補助金総合研究所R01GM086852、RC1CA145746（JAS）によってサポートされています。 JASは白血病リンパ腫協会の学者です。 JCは、がん研究所のア​​ーヴィング研究所フェロー。 MMは、国立科学財団助成統合大学院教育研究見習（NSF IGERT 0333389）によってサポートされています。

1。フルオロフォアとのDNAプローブのラベリング：ニック翻訳（〜6時間） <ol><li>クリーンBAC DNA（マキシプレップにより調製）またはプラスミドやPCR産物は、H 2 Oに再懸濁した全てが、標識に使用することができます注堅牢FISHの信号については、プローブは少なくとも10キロバイトに及ぶべきであること。 </li><li> 37℃（全ての試薬 ​​を表1に記載されてい?...

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上記の詳細テクニックは、リンパ球の30,31を開発中の免疫グロブリンおよびTCRA / D座のV（D）J組み換えの規制を分析するために私たちの研究室で使用されていた。我々は、この技術は、異なる細胞型では、様々な核タンパク質、核コンパートメントと遺伝子座の検出に適応させることができると確信しています。プロトコルの修正が必要になることがあり、この場合に集?...

エピジェネティックなメカニズムトリガ成立と発達と細胞型特異的転写プロファイルの継承。 1レベルでこれは、アクティブまたはサイレントゲノム領域を定義クロマチン包装の変調を伴う。大規模に、ゲノムと核構造のグローバル3D組織はまた、転写パターンの制御に役割を果たしています。したがって、これらのepigenomic機能の解剖は、遺伝子が6-11を規制されている方法を完全に理解するために不可欠です。 組み合わせた免疫蛍光法および3DのDNA FISHは、特異的な相互作用/核内のDNA配列および/またはタンパク質の関連を評価することによって、分子および生化学的解析を補完するユニークな機会を提供します。さらに、このようなクロマチン免疫沈降（ChIP-seq）が染色体またはキャプチャのコンフォメーションなどのゲノムワイドなハイスループット技術は深いシーケンシングと相まってしばらく（4C-SEQ、5Cは、Hi-C）にグローバルdatを提供細胞集団は12日 、免疫蛍光/ DNA FISHの技術は、単一細胞レベルでの解析を可能にします。 ここでは、3D顕微鏡や分析（3D免疫FISH）が続いたDNA FISHによって免疫蛍光法およびDNA配列によってヒストン修飾の同時検出のためのプロトコルを記述します。このプロトコルの利点は、DNAやタンパク質の構造の保全を組み合わせた視覚化したものです。この分野での我々の経験では、既存のプロトコルを改善し、簡素化することができるようになりました。我々は組換えを受けてリンパ球におけるDNA二本鎖切断を検出するには、このプロトコルを使用していたが、この方法は、他のタンパク質と他の細胞型に適用することができます。

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> H 2 O </td><td>フィッシャー</td><td> ＃BP2470 </td><td></td></tr><tr><td>アーゼ</td><td>シグマ</td><td> ＃R4642 </td><td></td></tr><tr><td>のdNTP </td><td>シグマ</td><td> ＃DNTP100 </td><td></td></tr><tr><td>アレクサdUTPを</td><td>インビトロジェン</td><td> C-11401に＃C11397 </td><td></td></tr><tr><td> Cy3またはCy5 dUTPを</td><td>フィッシャー</td><td> ＃45-001-XXX </td><td></td></tr><tr><td> DNase Iを</td><td>ロッシュ</td><td> ＃04536282001 </td><td></td></tr><tr><td> DNA Pol Iは</td><td>バイオラボズ</td><td> ＃M0209 </td><td></td></tr><tr><td> 0.025μmフィルター</td><td> Millipor電子</td><td> ＃VSWP02500 </td><td></td></tr><tr><td> COT-1 DNAを1 mg / mlの</td><td>インビトロジェン</td><td> ＃18440 </td><td></td></tr><tr><td> Hybloc DNAを1 mg / mlの</td><td>応用遺伝学</td><td> ＃MHB </td><td></td></tr><tr><td>サケ精子</td><td>シグマ</td><td> ＃D1626 </td><td>粉末をH 2 O中に10 mg / mlで再懸濁される</td></tr><tr><td> NAAC（酢酸ナトリウム、pH 5.2緩衝液） </td><td>シグマ</td><td> ＃S7899 </td><td></td></tr><tr><td>フィコール400（モルBIOLグレード） </td><td>フィッシャー</td><td>第525位</td><td></td></tr><tr><td>ポリビニルピロリドン（MOL BIOLグレード） </td><td>フィッシャー</td><td> ＃BP431 </td><td></td></tr><tr><td>デキストラン硫酸パウダー</td><td>シグマ</td><td> ＃D8906 </td><td></td></tr><tr><td> SSPE（生理食塩水リン酸ナトリウム、EDTA）を20倍ソリューション</td&gt; <td>フィッシャー</td><td> ＃BP1328 </td><td></td></tr><tr><td>ホルムアミド</td><td>フィッシャー</td><td> ＃BP227 </td><td></td></tr><tr><td>カバースリップ</td><td>フィッシャー</td><td> ＃12-548-B </td><td></td></tr><tr><td>スライド</td><td>フィッシャー</td><td> ＃12から550 </td><td></td></tr><tr><td> 6ウェルプレート</td><td>フィッシャー</td><td> ＃0720080 </td><td></td></tr><tr><td> 10×PBS、 </td><td>フィッシャー</td><td> ＃MT-46-013-CM </td><td></td></tr><tr><td>ポリ-L-リジン溶液</td><td>シグマ</td><td> ＃P8920 </td><td></td></tr><tr><td>パラホルムアルデヒド、プリル、95％ </td><td>シグマ</td><td> ＃441244 </td><td></td></tr><tr><td>トリトンX-100、MOL BIOLグレード</td><td>シグマ</td><td> ＃T8787 </td><td></td></tr><tr><td> BSA（ウシ血清アルブミン）フラクションV </td><td>フィッシャー</td><td> ＃BP 1600 </td><td></td></tr><tr><td>正常ヤギ血清</td><td>ベクトルラボ</td><td> ＃S-1000 </td><td></td></tr><tr><td> Tween-20を、モルBIOLグレード</td><td>シグマ</td><td> ＃P9416 </td><td></td></tr><tr><td> SSC（食塩クエン酸ナトリウム）20倍ソリューション</td><td>フィッシャー</td><td> ＃BP1325 </td><td></td></tr><tr><td> Gold退色防止試薬を延長</td><td>インビトロジェン</td><td> ＃P36930 </td><td></td></tr><tr><td> DAPI（4 &#39;、6 - ジアミジノ-2 - フェニルインドール） </td><td>シグマ</td><td> ＃D9542 </td><td></td></tr><tr><td>最高のテスト1コートラバーセメント</td><td>アートやオフィス用品店</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 表1に特異的な試薬及び小型機器。 </td></tr></tbody></table>

combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization

3D共焦点顕微鏡（3DのDNA FISH）を、続いて3次元的に保存核にDNAプローブを用いた蛍光 in situハイブリダイゼーションは、個々の細胞における遺伝子座の位置、サブ領域の染色体または全体の領土を視覚化するための最も直接的な方法を表しています。このタイプの分析は、核のグローバルアーキテクチャだけでなく、核空間内の特定のゲノム遺伝子座や地域の行動への洞察を提供します。免疫蛍光法は、他の一方で、核タンパク質の検出（修正ヒストン、ヒストンバリアントと修飾、転写機構や要因、原子力サブコンパートメントなど）を許可します。免疫蛍光法および3DのDNA FISHを組み合わせるにおける主要な課題は核の3Dアーキテクチャと同様に抗体によって検出されるエピトープを保持する一方であり、他方では、DNAプローブの浸透が検出できるように遺伝子座や染色体テリトリーの1月5日。ここでは、3D保存核内ゲノム遺伝子座とクロマチン修飾の可視化を組み合わせたプロトコルを提供します。

DNAと免疫FISHはBおよびTリンパ球の開発時に抗原受容体遺伝子座のV（D）J組み換えのプロセスに関連付けられている核の組織の変化を研究するためにSkokラボで使用されています。私に私たちを有効にする）遺伝子座の両端（収縮）の間の測定距離は上記の詳細技術ⅱ）対立遺伝子または遺伝子座の間の測定距離（ペアリング）、iii）は座、静脈内で発生するDNA損傷を解析するには）対立遺伝?...

Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

ここでは、3D顕微鏡や分析（3D免疫DNA FISH）が続いたDNA FISHによる蛍光抗体法およびDNA配列によるヒストン修飾の同時検出のためのプロトコルを記述します。

3D原子力組織の変化を研究するために3D保存状態間期核に組み合わさ免疫やDNA FISH

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

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Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

DNA-based Fish Species Identification Protocol

DNAベースの魚種の識別プロトコル

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Robust 3D DNA FISH Using Directly Labeled Probes

直接標識プローブを用いた堅牢な3DのDNA FISH

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

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

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

組み合わせたDNA-RNA蛍光<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）差別雌マウス胚性幹細胞におけるX染色体の不活性化を研究するために、

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

慢性後のマウス脾細胞におけるDNA損傷と修復の測定<em&gt;インビボ</em&gt;β-およびγ-放射線の非常に低い用量への暴露

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

ミトコンドリア局在と原子力細胞周期キナーゼ、Cdk1のの機能を研究するための実験的アプローチ

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

enChIPによって特定のゲノム領域と関連する分子の同定の単離

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

線虫の加齢とともに固有の蛋白質の集合の変化を研究するためのメソッド

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

凍結切除ゼブラフィッシュ胚に対する順次免疫蛍光と免疫組織化学

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...