我々は、このプロトコルの開発にDörtheユーリッヒ、ジェニファーラウンドとアンドリューマーラの貢献を感謝したい。 NICHD、米国癌学会とマーチオブダイムスが提供する研究支援。

1。固定<ol><li>一晩4胚℃でPBS中4％パラホルムアルデヒド（PFA）を持つ。を修正</li><li>削除修正と室温（RT）で2 × PBS、5分ごとに洗う。 </li><li>時計職人の鉗子を使用してガラスのうつ病のプレートにPBSで手動でdechorionate胚。 dechorionationした後、胚は彼らがポリプロピレンのピペットに付着可能性があるため、ファイアーポリッシュガラスパスツールピペットを用いて転送されます。 </li><li> 25％、50％と各5分間、PBS中の75％メタノール、一連の胚を移す。 </li><li>新鮮なメタノールで交換して、100％メタノールで液を交換して5分間インキュベートし。 </li><li>一時間の最小値まで-20℃で場所は胚。 （私たちは、通常は一晩胚をインキュベートする。situハイブリダイゼーションの標準1年以上保存されている胚で動作しますが、我々はin situハイブリダイゼーション蛍光灯の高解像度が長期保管によって失われているかどうかを検討していない。） </li><li> 75％、50％、室温でPBSTで25％メタノールで5分間ごとに胚を洗浄してください。室温で5分間PBSTで各2回洗浄する。 </li><li>室温でPBS中4％PFAで20分間再修正。 </li><li>室温で5分間PBSTで各2回洗浄する。 </li></ol> PFAに関する注意： 我々は（試薬の表を参照）-20℃でアリコートで4％PFAを格納する。初期固定のために、我々は以前に解凍されたことがないPFAを使用してください。その後の注視のために、我々は多くの場合、それ以前に解凍されたPFAを使用してください。初期固定は、このプロトコルで成功する染色ために重要であると表示されます。 2。プロテイナーゼとかん流後<ol><li>胚を透過性に3〜12分間室温でプロテイナーゼK（PBSTで5μg/ml）とダイジェスト。 （インキュベーション時間は、若い段階ではより敏感であるため、胚の年齢に依存する。また、酵素のバッチ処理に依存する。）体節形成期胚では、我々は通常、3〜4分のために透過性。このインキュベーションの間、我々は、その側のマイクロ遠心チューブを置く。 </li><li> PBSTで簡単にすすぎ、PBSTで5分間一回洗浄。 </li><li>室温でPBS中4％PFAで20分間再修正。 </li><li>室温でPBSTで、各5分間、2回洗浄する。 </li></ol> 3。プレハイブリダイゼーション<ol><li>で65℃で5分間胚をインキュベートHYB -。 </li><li> HYBで少なくとも1時間、65℃でプレハイブリダイゼーション+。 </li></ol> 4。ハイブリダイゼーション<ol><li> preHYBのすべてが、50μlを削除するが、完全に水没胚を維持することを確認してください。 </li><li>軽くチューブをタッピングし胚と混合する各リボプローブの1〜2μL（ジゴキシゲニン-およびフルオレセイン標識リボプローブ）を追加します。プローブの量は、典型的には20μlのプローブの合成反応から1 -2μlのです。 </li><li>フルオレセインが光に敏感であることを考えると、チューブがアルミホイルに包まれたり、そうでなければこれ以降、最小限の光にさらされるべきである。 </li><li> 65 º Cで一晩胚をインキュベート</li></ol> 5。プローブの取外しこの時点で前方に不足の洗剤からソリューションがあることに注意してください。洗剤の除去は、染色反応を助けるように見えますが、胚はかなり粘着性になることがありません。 <ol><li>リボプローブを取り外します。 </li><li> 50％のformamide/2xSSCで65℃で2 × 30分間洗浄します。 </li><li> 2 × SSCで65℃で15分間洗浄する。 </li><li> 0.2 × SSCで65℃30分間洗浄する。 </li></ol> 6。抗フルオレセイン抗体のインキュベーション<ol><li>ブロッキング試薬1Xマレイン酸緩衝液を加えた2％溶液を500μLで室温にて少なくとも1時間ブロック（試薬の表を参照）。 </li><li>ブロッキング溶液で1:500に希釈したとしてロシュから提供される抗フルオレセイン- POD抗体を、追加します。 </li><li> 4℃で一晩インキュベートするこのインキュベーションの間、我々は、その側のマイクロ遠心チューブを置く。 </li><li> 1Xマレイン酸緩衝液で4 × 20分間洗浄します。 PBSで2回（各5分間）洗浄する。 </li></ol> 7。フルオレセインラベル付きプローブの検出<ol><li> TSA Plusのフルオレセイン溶液で30〜60分インキュベートする。 （染色液を作る前に、TSAの基板にスピンダウン。反応は、パーキンエルマーの増幅の希釈バッファーでチラミド試薬1:50に希釈が。）このインキュベーションの間、我々は、マイクロ遠心チューブは横に置く。反応時間は、各プローブに対して経験的に決定する必要があります。残念ながら、染色反応は基質が蛍光であるとして視覚的に監視することができない、と一染色反応を通してユビキタス緑色蛍光が表示されます。 </li><li> 30％、50％、75％、PBSで100％メタノールで10分間ずつ洗浄する。 </li><li>第一ペルオキシダーゼを不活性化するために30分間、メタノールで1％の溶液H 2 0 2でインキュベートする。 </li><li> 10分各75％、50％、PBSで30％のメタノールを洗ってください。その後、PBSで10分間ずつ2回洗浄する。 METのそのすべてが重要です。hanolは削除される。 </li></ol>チラミドシグナル増幅について​​の注意： 我々は、パーキンエルマーTSAキットを使用している。我々はAlexa -チラミド基質がパーキンエルマーの増幅の希釈バッファーを使用しても汚れがわかりますが、我々はInvitrogen社/ Molecular Probes社のキットに付属の染色バッファーを使用して成功を収めていない。最後に、我々は、フルオレセインとAlexa - 647は影響を受けないままCy5蛍光は、その後のメタノール/ H 2 O 2処理によって除去されることを見出した。 Cy3にも悪影響を、それが構造的にCy5標識に関連しているとして、メタノール/ H 2 O 2処理によって影響を受ける可能性があります。このような理由から、Cy3およびCy5 TSA反応は、その場のプロトコルで二重蛍光第二染色反応に使用する必要があります。 8。 ANTI -ジゴキシゲニン抗体インキュベーション<ol><li> 1Xマレイン酸緩衝液を加えた2パーセントのブロッキング試薬の溶液に、室温で少なくとも1時間再び胚をブロックします。 </li><li>上記ブロッキング溶液で1:1000希釈でRoche社によって提供された抗DIG POD抗体を追加</li><li> 4℃で一晩インキュベートするこのインキュベーションの間、我々は、その側のマイクロ遠心チューブを置く。 </li><li> 1Xマレイン酸緩衝液で4 × 20分間洗浄します。 PBSで2回（各5分間）洗浄する。 </li></ol> 9。ジゴキシゲニン-ラベル付きプローブの検出<ol><li>このインキュベーションの間にTSAで30〜60分プラスCy5とソリューションを（染色液を作る前に、TSAの基板にスピンダウン。反応は、増幅の希釈バッファーでチラミド試薬1:50に希釈）インキュベート、我々は、マイクロ遠心チューブは横に置く。反応時間は、各プローブに対して経験的に決定する必要があります。 </li><li> PBSTで10分間ずつ3回洗浄する。 </li></ol> 10。ヨウ化プロピジウム染色<ol><li> 2 × SSCで2回（各5分間）洗浄する。 </li><li> 37℃で30分間胚をインキュベートします10μlのRNアーゼ（100μg/mlの最終濃度）と50μlの2 × SSCTでC。 </li><li>室温で2 × SSCで3分間ずつ6回洗浄する。 </li><li> 2 × SSCで330μg/mlのヨウ化プロピジウムの溶液中で8​​分間胚を染色。 </li><li>室温で2 × SSCで3分間ずつ6回洗浄する。 </li><li>室温でPBS中4％PFAで20分間固定する。 </li><li>室温でPBSTで、各5分間、2回洗浄する。 </li></ol> 11。拭き取り方<ol><li> 10分25％それぞれPBSTで50％グリセロールのために胚をインキュベートする。 4℃で75％グリセロール中で一晩クリア</li><li>我々は、胚および顕微鏡スライド上にマウントフラットを細かく分析し、deyolk。卵黄はかなりのバックグラウンド蛍光を生成することができます。解剖では、胚がgylcerolでovenightをクリアした後にはるかに簡単です。 </li></ol>ソリューション： <table border="1"><tbody><tr><td> PBST </td><td> PBS +0.1％のTween </td><td></td></tr><tr><td> SSCT </td><td> SSC +0.1％のTween </td><td></td></tr><tr><td> HYB - </td><td> 50％のホルムアミド 5xSSC 0.1％のTween - 20 </td><td></td></tr><tr><td> HYB + </td><td> HYB - 5mg/mlトルラ（酵母）RNA 50μg/mlヘパリン</td><td style="width: 300px;">トルラRNAは、その後のフェノール、とRNAのプロテイナーゼK消化によって調製されるフェノール - クロロホルム - 、及びクロロホルム- extraction.The RNAを沈殿させ、DEPC処理水に溶解する。 </td></tr><tr><td> 1 ×マレイン酸バッファー</td><td> 150mmのマレイン酸、100mMの塩化ナトリウム液（pH7.5） </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="図1" width="600" height="688" /> 図1代表的なin situハイブリダイゼーション二重蛍光全体のマウントの結果を。 （AC）。画像は10体節段階で、ゼブラフィッシュ胚の後部領域を介して単一の共焦点セクションを示しています。ビューには、上部に前方と背側です。ヨウ化プロピジウムで染色された核は青色です。 （A）deltaCのmRNAは、ジゴキシゲニン標識リボプローブとTSA - Cy5の試薬 ​​を用いて検出した。 （B）HER1の遺伝子から転写されたmRNAフルオレセイン標識リボプローブとTSA -フルオレセイン試薬で検出された。 （C）緑と赤のチャネルを統合すると、明確に二つの遺伝子の異なるまたは重複する遺伝子発現の領域を識別します。 （D、E）HER1表現の詳細は、この手順の高解像度を実証。 mRNAの細胞内局在を明確に識別できる。矢頭は核内染色のドットによって明らかにアクティブな転写を示す細胞を、示している。矢印は、mRNAの細胞質局在を示す細胞を示している。 （F、G）HER1とdeltaCのための二重染色は、別々に両方の遺伝子（白矢印）、またはいずれかの遺伝子を転写している細胞（赤と緑の矢印）を明らかにする。

<ol>
	<li>Schulte-Merker, S., Ho, R. K., Herrmann, B. G., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+product+of+the+zebrafish+homologue+of+the+mouse+T-gene+is+expressed+in+nuclie+of+the+germ+ring+and+the+notochord+of+the+early+embryo.">The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclie of the germ ring and the notochord of the early embryo.</a> Development. 116, 1021-1027 (1992).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23, 345-358 (2001).</li><li>J&#252;lich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Mara, A., Schroeder, J., Chalouni, C., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming,+Initiation+and+Synchronization+of+the+Segmentation+Clock+by+deltaD+and+deltaC.">Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC.</a> Nat Cell Biol. 9, 523-530 (2007).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li></ol>

ここで紹介するプロトコールは、典型的なアルカリホスファターゼを介した反応で30〜45分のために染色した後、クリーンな強力な信号を与えるプローブでうまく動作します。 in situハイブリダイゼーションのプロトコールで蛍光を実行する前に、我々は常に標準の非蛍光プロトコルを（プローブの合成プロトコルと共に補足資料で提供される）を使用して私たちのプローブをテストします。?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Paraformaldehyde 16% solution, EM Grade</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Proteinase K, recombinant PCR grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115879001</td>
<td>Make a 20mg/ml stock solution in water. Store in 1ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Blocking Reagent</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11096176001</td>
<td>Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at &#x2013;20&deg;C. Stable over multiple freeze/thaws.</td>
</tr>
<tr>
<td>Anti-Fluorescein-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426346910</td>
<td>Aliquot and store at -20&deg;C. Keep one working aliquot at 4&deg;C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11207739910</td>
<td>As above.</td>
</tr>
<tr>
<td>TSA Plus Fluorescein system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL741001KT</td>
<td>Prepare each vial of TSA reagent only when needed. Dissolve in 60&mu;l DMSO. Store at 4&deg;C.</td>
</tr>
<tr>
<td>TSA Plus Cyanine5 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL745001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Plus Cyanine3 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL744001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>T20926</td>
<td>Dissolve tyramide reagent in 150&mu;l DMSO, aliquot and store at &#x2013;20&deg;C.</td>
</tr>
<tr>
<td>RNase, DNase free</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11119915001</td>
<td>Supplied as 500&mu;g/ml solution</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>P-3566</td>
<td>Supplied as 1mg/ml solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish whole mount high-resolution double fluorescent in situ hybridization

全体のマウント<em&gt;その場で</em&gt;ハイブリダイゼーションは、発生生物学で最も広く使用されている技術の一つです。ここで、我々は、高解像度の二重蛍光を示す<em&gt;その場で</em単一の遺伝子の正確な発現パターンを解析するためのと二つの遺伝子の発現領域の重なりを判定するための&gt;ハイブリダイゼーションプロトコル。プロトコルは、標準の修正バージョンです。<em&gt;その場で</emこのようなNBT / BCIPやFast Redなどのアルカリホスファターゼと基板を使用して&gt;ハイブリダイゼーション<sup&gt; 1,2</sup&gt;。このプロトコルは、チラミドシグナル増幅（TSA）と一緒に標準的なジゴキシゲニンおよびフルオレセイン標識プローブを利用<sup&gt; 3</sup&gt;。市販のTSAキットには、緑から遠赤色に蛍光発光のような柔軟な実験計画がこのようなヨウ化プロピジウム、またはタンパク質の蛍光免疫組織化学などの様々な核染色と組み合わせて使用​​することができることができます。 TSAは、迅速に共有結合標識アンチセンスリボプローブのすぐ近くに、部分、通常はチロシン残基に結合する反応性蛍光基質を生成します。結果として得られる染色パターンは、mRNAの細胞内局在性の高分解能であるレーザ走査共焦点顕微鏡を用いて観察することができます。<sup&gt; 3,4</sup&gt;。一つは、染色体の遺伝子座での新生転写物を観察し、核と細胞質染色を区別し、mRNAの皮質局在などの他のパタ​​ーンを視覚化することができます。の研究<em&gt;ショウジョウバエ</em&gt; mRNAの約70％は頻繁にコードされたタンパク質の機能と相関する細胞内局在性の特定のパターンを示すことを示している<sup&gt; 5</sup&gt;。 3D共焦点データセットのコンピュータ支援復興と組み合わせると、我々のプロトコルは、全体の脊椎動物の胚では、サブ携帯の解像度を持つmRNAの分布を詳細に分析することができます。

Watch this Scientific Journal Video about Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization at JoVE.com

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

in situハイブリダイゼーション全体のマウントは、発生生物学で最も広く使用されている技術の一つです。ここで、我々は、単一の遺伝子の正確な発現パターンを解析するためと、二つの遺伝子の発現領域の重なりを判定するためのin situハイブリダイゼーションのプロトコールで二重蛍光の高解像度を示す。我々は、組織の組織を強調するためにカウンター染色核のヨウ化プロピジウムを含む。

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 ...

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Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.2K ビュー.  Yale University. in situハイブリダイゼーション全体のマウントは、発生生物学で最も広く使用されている技術の一つです。ここで、我々は、単一の遺伝子の正確な発現パターンを解析するためと、二つの遺伝子の発現領域の重なりを判定するためのin situハイブリダイゼーションのプロトコールで二重蛍光の高解像度を示す。我々は、組織の組織を強調するためにカウンタ...

ビデオ: Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

全マウントを使用するその場で</em分子とOrganismal生物学をリンクする>ハイブリダイゼーション

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

の蛍光タンパク質の偏転アフリカツメガエルWntシグナルへの対応に>外胚葉

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全体のマウント<em&gt;上皮内</emE11.5マウス胚へのE8.5&gt;ハイブリダイゼーション

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解像度全マウント<em&gt;その場で</em遺伝子発現と機能を研究するためにゼブラフィッシュ胚内&gt;ハイブリダイゼーション

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Methods Article

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