We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1.プローブの準備<ol><li> 5&#39;-アミノ修飾を有する複数の固有のXistにおけるオリゴヌクレオチド（20〜30ヌクレオチド長のオリゴヌクレオチド、63-65ºCの融解温度、 表1）を取得し、水に一時停止。 </li><li> 5&#39;-アミ​​ノ修正（合計4.5μgの）、製造業者の指示以下のアミン反応性蛍光色素で標識を用いてオリゴヌクレオチドのプール等モル量。 <ol><li>ヌクレアーゼを含まない水の最終5μlのプールされたオリゴヌクレオチドを​​溶解し、オリゴヌクレオチド溶液に、1M重炭酸ナトリウムの3μlを添加する。 </li><li>簡単にアミン反応性染料と渦のバイアルに2μlのDMSOを追加します。 </li><li> DMSO中の反応性染料をオリゴヌクレオチド溶液を混合し、暗所で1時間室温で混合物をインキュベートする。 </li></ol></li><li>エタノール沈殿に続くG-25カラムで蛍光標識されたオリゴヌクレオチドプローブを精製する。 <ol><li過剰保存バッファーを除去するために1分間750×gで&gt;遠心機G-25カラム。 </li><li>ステップ1.2.3で生産ラベリング混合物に40μlのヌクレアーゼフリー水を追加し、G-25カラムに適用されます。 2分間750×gで遠心分離する。 </li><li>ステップ1.3.2から精製されたプローブを、50μlのヌクレアーゼフリー水、10μlの3M酢酸ナトリウム（pH5.2）および250μlのエタノールを加える。 4℃で15分間21000×gで30分間の遠心のため-80℃で保存。 </li><li> 70％エタノール1mlで一回沈殿させたオリゴヌクレオチドを​​洗ってください。 </li><li> 50μl中に再懸濁ヌクレアーゼフリー水またはTE（10mMのトリス-HCl、pH7.5; 1mMのEDTA）、約10μMの最終濃度。 </li></ol></li><li>ハイブリダイゼーション緩衝液（; 2×SSC [300mMのNaCl、30mMのクエン酸ナトリウム]; 2mg / mlのBSA、10％硫酸デキストラン、10％ホルムアミド）を用いて250nMでの最終濃度に蛍光標識されたオリゴヌクレオチドプローブを希釈する。 注：このクイックFISHプロトコルで、オリゴヌクレオチドPXistの中のローブを設計し、ハイブリダイゼーションのためのインキュベーション時間を短縮するためにオリゴヌクレオチドプローブを使用して、通常のFISHプロトコルよりも高い濃度で使用した。 </li></ol> 2.スライドさせて準備注：スライドをマウス胚性幹（ES）または胚様体（EB）を用いた免疫FISH用に調製することができるポリ-リジンで処理したスライド上に直接成長または細胞懸濁液をサイトスピンを用いて、セル20、21を微分する。ここで、サイトスピンによりスライドの準備を示している。 <ol><li> 6ウェルディッシュ中の培地を吸引除去する。 PBSで洗浄し（室温）、そして徹底的に吸引除去する。 </li><li> 37℃で5〜10分間インキュベート、6ウェルディッシュにトリプシンの0.5ミリリットルを追加します。 </li><li>培地の5ミリリットルを加え、穏やかに数回ピペッティングして細胞を分散させる。 </li><li>室温で200×gで5分間、15mlチューブに遠心し、細胞を移す。 </li><li>メディアのANを破棄優しく2mlのPBSで細胞を懸濁dは。 </li><li>血球計数器を用いて細胞をカウントし、PBSによる4×10 5細胞/ mlで細胞懸濁液を希釈する。 </li><li>クリップでスライド、フィルタカードとチャンバーを組み立て、サイトスピンのローターで適切なスロットに入れます。サイトスピンで組み立てられたユニットの各チャンバーにステップ2.6で調製した各サンプルを250μlを追加。室温で10分間、1500rpmで遠心する。 </li><li> 40ミリリットルの氷冷PBS、CSKバッファーのそれぞれと氷上で3コプリンジャーフィル（10mMのPIPES、pHが6.8、100mMのNaCl、300mMのスクロース; 3のMgCl 2）、およびCSKTバッファー（0.5％トリトンとCSKバッファーX-100）、それぞれ。 </li><li>サイトスピンが終了した後、すぐに氷冷したPBS中にスライドを転送し、5分間インキュベートする。 </li><li>氷のように冷たいCSKバッファにスライドを移し、1分間インキュベートする。 </li><li>氷のように冷たいCSKTバッファにスライドを移し、5分間インキュベートする。 </li><li>バック氷のように冷たいCSK bufにスライド移動する。FERと1分間インキュベートする。 </li><li> PBS中の4％パラホルムアルデヒド中にスライドを移し、室温で10分間インキュベートする。 </li></ol> 3.免疫蛍光<ol><li>いくつかの秒（0.1％のTween-20を1×PBS）PBSTでコプリンジャーにスライドを置きます。 </li><li>残りのバッファがスライドダウン流れることを可能に斜めにスライドを置きます。慎重にスライドから余分な液体を拭き取り、水と空のピペットチップボックスに入れてください。静かにカバースリップを置き、ブロッキング溶液を40μl（1×PBS、1％BSA、0.1％のTween-20）を用いてスライドをオーバーレイし、室温で10分間インキュベートする。 </li><li>カバースリップを外し、ブロッキング溶液を除去し、1の濃度に希釈し、ヒストンH3K27me3一次抗体40μlの追加：ブロッキング溶液で500を。バックスライド上にカバースリップを置き、室温で30分間インキュベートする。 注：ブロッキング溶液中のRNase阻害剤は、このRNA FIS中のRNAを保存するために役立つかもしれないポリクローナル抗体が使用されているHプロトコル。 </li><li>ウォッシュを優しく洗いながら揺れ、5分ごとに、PBSTで満たされたコプリンジャーに2回スライドさせます。 </li><li>残りのバッファがスライドダウン流れることを可能に斜めにスライドを置きます。慎重に余分な液体を拭き取り、バック先端ボックスにスライドを置く。二次抗体（1：500）の40μlのオーバーレイブロッキング溶液中で希釈し、カバースリップを置き、暗所で15分間インキュベートする。以下の手順については、すべての回で暗闇でスライドを保つ。 </li><li>ステップ3.4のように、PBSTでjarをコプリンで2回洗浄する。 </li></ol>オリゴヌクレオチドプローブ4. RNA FISH <ol><li>ステップ4.3でのRNA FISH 37℃での乾燥からスライドを防ぐために、下部の水と空のピペットチップボックスを事前に温める。 </li><li>残りのバッファがスライドダウン流れることを可能に斜めにスライドを置きます。慎重にステップ3.6からスライドから余分な液体を拭き、ワットとピペットチップボックスにスライドを置く下部のER。スライド上のFISH洗浄緩衝液（2×SSCで10％ホルムアミド）の500μLを加え、室温で5分間インキュベートする。 </li><li> FISH洗浄溶液を外し、スライド上にオリゴヌクレオチドプローブの8μlを添加、カバースリップを配置し、下部の水と予め温めピペットチップボックスにスライドを転送する。 1〜2時間、37℃でインキュベートする。 </li><li> FISH洗浄緩衝液、DAPIによるFISH洗浄緩衝液、および2×SSCで37℃の水浴中で3予め温めコプリンジャーを置き。 </li><li> RNA FISH 1-2時間インキュベートした後、予め温めFISH洗浄緩衝液中にスライドを置く。 </li><li>カバースリップ、スライドの外に低下するまで数分間スライドをインキュベートします。 </li><li>バック洗浄バッファーにスライドを置き、5分間のインキュベーションを続ける。 </li><li> 0.5 / mlのDAPIで予め温めFISH洗浄緩衝液で別のコプリンジャーにスライドを移し、5分間インキュベートする。 </li><li>予め温め2X SSCで別のコプリンジャーにスライドを移し、5マイルインキュベートN。 </li><li>残りのバッファがスライドダウン流れることを可能に斜めにスライドを置きます。慎重に余分な液体を拭き取り、乾いた先端ボックスにバックスライドを置く。スライド上にDAPIで退色防止の4μLを追加し、カバースリップを配置。 </li><li>マニキュアでカバースリップを密封し、顕微鏡下で観察する。スライドは、数ヶ月間暗所で4℃で保存することができる。 </li></ol>

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No conflicts of interest declared.

本稿では、H3K27me3ためのスライドの準備、XistのRNA FISH、および免疫蛍光を完了するために、5未満の時間がかかる迅速な免疫FISHプロトコルを提示している。通常RNA FISHのための一晩のインキュベーションを必要とするXist RNA検出のための一般的な免疫FISH法と比較して、このプロトコルは、有意に費やされる時間を減少させるだけでなく、オリゴヌクレオチドプローブを使用して免疫FISHの感度を...

哺乳類のX染色体不活性化（XCI）は、雌では二つのX染色体の1が1転写不活性化され、XXとXY間のX連鎖遺伝子量のアンバランスを補償するための戦略である。 X染色体不活性化は、長い非コードRNA（lncRNA）研究のための優れたモデルシステムです。 X染色体不活性化は、複数のlncRNAsによって調節され、広範にlncRNAs、転写、クロマチン構造および核組織2,3間のクロストークメカニズムを解明するために、過去数十年にわたって研究されてきた。 X染色体上に位置するX不活性化センター（XIC）は、非コードRNA 4を産生する遺伝子の数で構成される複雑な遺伝子座である。 X-非アクティブな特定の転写産物（のXist）lncRNA真獣類哺乳動物においては、X染色体不活性化5,6に重要な役割を果たしているそのようなlncRNAです。 Xistの転写物のfuの位置を囲むトゥーレXiはX染色体不活性化を開始し、RNA FISHを用いて可視化するとき雲のように表示される。この形成は、「Xistのクラウド」7と呼ばれている。のXist RNAは、様々なクロマチン修飾酵素と相互作用するので、サイレントクロマチンと抑圧的な転写のための別のエピジェネティック修飾とXistの雲の共局在は、X染色体不活性化8の間に観察されている。例えば、XistのRNAはH3K27me3を担当し、抑圧的なクロマチン状態9を誘導するポリコーム抑制複合体2（PRC2）と対話します。 Xiと集中的なH3K27me3の修正との共局在上のXistの雲の発生は、西10,11の通性ヘテロクロマチン風景を表している。 そのようなDNA / RNA FISHなどの細胞遺伝学的技術は、強化された感度で最近、高度な技術を放射性標識したプローブ12を用いた従来の方法から長い道のりを歩んでいると複数のオリゴヌクレオチドプローブ13,14を用いた蛍光イメージング。免疫蛍光と連結DNA / RNAのFISHは、日常的に、時空間核組織、RNAの局在化、クロマチン構造および修正を理解するために、細胞学的ツールとして使用されてきた。 RNA FISHのための最も標準的なプローブの調製は、プラスミドまたは細菌人工染色体（BAC）クローンおよびニックトランスレーションまたはランダムプライミング15のいずれかそれらのその後の標識の使用を含む。しかし、ヒトにおけるマウスの遺伝子の約70％の遺伝子の40％は、したがって、センスおよびアンチセンス転写物を区別するために、鎖特異的FISH法を必要とする、センスおよびアンチセンス転写物16の重なりを示している。 インビトロ転写されたRNAプローブ（リボプローブ）は、多くの場合、鎖特異的RNA FISH 17,18のために使用される。しかしながら、これは、T7、SP6又はT3プロモータープラスミドクローンまたはPCR産物を調製し、リボプローブを合成することを含む。さらに、リボプローブはGENOMから派生icをDNAまたはcDNAは、多くの場合、高いバックグラウンドノイズを生じる​​非特異的領域および反復エレメントを含む。別の問題は、長さが数百ヌクレオチドであり、複数のフルオロフォアを含むリボプローブは、効率的に核内に浸透することができないということである。これを回避するために、終了時の単一のフルオロフォアで標識された複数の短いオリゴヌクレオチドプローブは、良好な感度、均一な信号強度、精製および取り扱い14の容易さを有するように開発されてきた。また、DNAオリゴヌクレオチドは、一般的にRNAよりも安定である。我々は、X染色体不活性化の過程のXist lncRNAによって誘発される西のエピジェネティックなダイナミクスを理解するために、免疫蛍光でのXist RNA FISH 19のオリゴヌクレオチドを用いて、同様の戦略を適用した。このプロトコルは、オリゴヌクレオチドプローブおよび適切な細胞の調製、ならびに免疫蛍光およびRNA FISHの利用の作成を記載している。複数oligonucleを使用してのXist RNA FISHotidesのXist RNA FISHは自分の研究室で日常的に行われた場合、長期的に費用対効果の高いアプローチです。この技術は、同時にエピジェネティック修飾または因子との共局在をマッピングしながら、細胞内でlncRNAsを同定するために用いることができる。プロトコルの1つの主な利点は、簡単に自分の研究関心に合わせてそれを変更する機能です。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
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quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

免疫蛍光（免疫FISH）、 インサイチュハイブリダイゼーション （FISH） で RNA蛍光を組み合わせることにより、タンパク質の局在に同時洞察、エピジェネティックな改変およびその他の詳細とRNAの局在化の空間的なダイナミックスを検出するために、単一細胞レベルで使用することができる技術を作成その免疫蛍光によって強調することができる。 X染色体不活性化は、長い非コードRNA（lncRNA）媒介遺伝子サイレンシングのためのパラダイムである。哺乳類の雌の二つのX染色体の1のX非アクティブな特定の転写産物（Xistの雲と呼ばれる）（のXist）lncRNAの蓄積は、X染色体不活性化を開始する重要なステップです。のXist RNAは、直接または間接的に、様々なクロマチン修飾酵素と相互作用し、不活性X染色体（XI）に明確なエピジェネティックな風景を紹介します。 xiの一つの既知のエピジェネティックな特徴は、ヒストンH3のトリメチルリジン27（H3K27me3）変形例である。ここでは、簡単かつ迅速免疫FISHを記述のXist RNAおよび関連するエピジェネティック修飾の局在を調べるためにH3K27me3の免疫蛍光に結合された複数のオリゴヌクレオチドプローブとのRNA FISHを使用してのXist RNAを検出するためのプロトコル。 インビトロ転写されたRNAプローブ（リボプローブ）と比較して、より短いインキュベーション時間とのXist RNAのより高感度な検出オリゴヌクレオチドプローブの結果を使用して。このプロトコルは、lncRNAsのダイナミクスとそれに関連するエピジェネティックな修飾、クロマチン構造、原子力組織と転写調節を理解するための強力なツールを提供します。

迅速な免疫FISHの代表的な画像を図1Aに示されている。 Xiの上のXist RNAクラウドとH3K27me3信号の共局在は、女性の細胞を分化で検出された。分化の際に12日目に、EB細胞の90％以上は、Xistの雲（ 図1B）を有していた。短いオリゴヌクレオチドプローブを効率のXist RNA（; 97％はn = 150、図1C）と共局在し、ほぼすべてのH3K27me3信号の可視化をもたらす、核内に侵入...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

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

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

クイック蛍光<em&gt;その場で</emX染色体不活性化におけるヒストン修飾の免疫蛍光とのXist RNA複合のための&gt;ハイブリダイゼーションプロトコル

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

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

13.8K ビュー.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

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

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.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性その場で</emノルウェースプルースと植物種の範囲に適用>ハイブリダイゼーションプロトコール

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

ゼブラフィッシュの全マウント高分解能二重蛍光上皮内ハイブリダイゼーション

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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.

蛍光その場で</emヒト精子の>ハイブリダイゼーション（FISH）プロトコル

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of 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.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNAの蛍光による単一細胞遺伝子転写の解析<em&gt; in situで</em&gt;ハイブリダイゼーション（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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.

蛍光灯と組み合わせることで染色体の複製タイミング<em&gt;その場</em&gt;ハイブリダイゼーション

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.

ホールマウントRNAを蛍光<em&gt;その場で</emの&gt;ハイブリダイゼーション<em&gt;ショウジョウバエ</em&gt;胚

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

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNAを<em&gt;その場</emホールマウント胚および細胞組織学で&gt;ハイブリダイゼーションは、海洋軟骨魚類に適合した

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...