著者は、この原稿の準備に関する重要な読書と有益な提案のためにSIバーンスタイン（サンディエゴ州立大学）を感謝。この作品は、SIバーンスタイン、SDSU、へとR. Bodmer、BIMRにNIHの補助金によって支えられて、とアメリカ心臓協会の西部米国アフィリエイトからG.フォーグラーへとA. Cammaratoにポストドクトラルフェローシップによる。

あなたが始める前に<ol><li>以下のソリューションを準備します。 <ol class="lalpha"><li>リラックスバッファー（10 mM EGTAを含む人工的なショウジョウバエの体液（ADH）（&quot; ショウジョウバエの心臓の鼓動を可視化する&quot;を参照）） </li><li>固定液（1 × PBS中4％ホルムアルデヒド） </li><li> PBSTx（PBS、0.1％トリトン- X - 100を含む） </li><li> PBSTxで適切に希釈した一次および種特異的蛍光標識二次抗体</li></ol></li><li>心臓の管（大人ハエおよび/ ​​または幼虫の）次のことを公開するショウジョウバエを解剖する<ol class="lalpha"><li> &quot; ショウジョウバエの心臓の鼓動を可視化&quot;のセミ無傷ショウジョウバエの心臓標本のプロトコル、または</li><li>次の変更とショウジョウバエの幼虫NMJ解剖プロトコル 1：使用すると、幼虫の解剖時にADHを酸素化し、腹側正中線から少し後ピンに置き換える。幼虫は腹側正中線に沿って切断されています。固定前に任意の組織を削除しないでください。 </li></ol></li></ol>蛍光染色<ol><li>すべての心がリズミカルに酸素ADHに拍動していることを確認してください。すぐにリラックスできるバッファとADHを交換してください。収縮が抑制されるようにそれぞれの心臓のチューブを調べます。 </li><li>固定液でリラックスしたバッファを置き換えることによって、心を修正。穏やかに振盪しながら室温で20分間インキュベートする。 （幼虫の準備の揺れには任意のステップで必要ではない、と心臓組織の整合性に悪影響を及ぼすと考えられる）。 </li><li>継続的に振とうしながら室温で試料をPBSTxで10分間3回洗浄する。 </li><li>大人のための、慎重に戻ってどのような残っていることがより楕円と少ない丸みになるような腹部のクチクラの腹側縁をトリミング。さらに、腹部と胸部の間にシングルカット、慎重にそしてきれいに別体のセグメントの両方とで心臓の前方領域に最小限のダメージを確認してください。幼虫の心のために、慎重に微細な鉗子を用いて体脂肪を取り除く。幼虫の心臓が特に壊れやすいので、他の筋肉や結合組織からはほとんどサポートしているので、脂肪除去は、細心の注意を払って実行する必要があります。これは心臓に損傷を与える可能性があるとして、気管枝を削除しないでください。 </li><li> PBSTxで希釈した一次抗体の50〜100μlを含むウェルの96ウェルプレートに、中心部に位置する心臓のチューブとの接触を避け、エッジで腹部クチクラのトリミング背部を転送します。ウェルあたり12以上の試料を置きます。継続的に攪拌しながら2時間室温でインキュベートする。インキュベーションをさらに4℃で一晩行うことができます℃の</li><li>一次抗体溶液を除去します。継続的に撹拌しながら室温で100μlのPBSTxで10分のために心を3回洗浄する。 </li><li>最後の洗浄緩衝液の除去に続いて、Alexa594 -ファロイジン（1:1000）を添加したPBSTxの二次抗体を100μlを、追加してください。室温で継続的に振とうしながら一時間インキュベートする。漂白蛍光団を防ぐためにカバーのサンプルを保管してください。 </li><li>二次インキュベーションに続いて、継続的に振とうしながら室温でPBSTx 100μlの10分のために心を3回洗浄する。洗浄ステップを通してカバーのサンプルを保管してください。 </li><li>トリトン- X - 100の除去については、10分間PBSを100μlの心の最後の時間をすすいでください。標本は、マウントする前に数日間、4℃で暗所に保存することができます。 </li></ol>アダルトハーツのために取り付け<ol><li> Vectashieldマウンティング培地の10μlを、顕微鏡のスライドに2つの18 × 18 mmのカバーガラスを付着する。カバースリップは〜10〜15ミリメートル離れて配置されるべきである。で、間に2つのカバースリップメディアをマウントするの小規模な3番目のドロップを置きます。 </li><li>第三カバーの中央にVectashield20μlを置きます。 </li><li>注意深くクチクラの極端な端をPBSの洗浄溶液から心のそれぞれを削除し、ゆっくりと第三カバースリップで封入剤のドロップにドラッグ心臓側を下に置きます。カバースリップ上のドロップあたり5つまで心を置きます。 </li><li>すべての心が下向きにしていることを確認するために顕微鏡下で確認してください。 </li><li>慎重に心を含んでいるカバーを反転し、すぐにそのようなカバーガラスのペアの間にVectashieldの液滴と心臓の管が付いている液滴は、ヒューズそのカバースリップのペアを含むスライドの上に置きます。 &quot;ブリッジは&quot;カバースリップによって形成されるべきである。心は、カバースリップと顕微鏡用スライドの間で中断されます。 </li><li>心は今上向きに直面していることを保証するために顕微鏡下で確認してください。 </li><li>マニキュアとの縁でカバーグラスを修正。 </li><li>心は今蛍光または共焦点顕微鏡を介してイメージを作成する準備が整いました。 </li></ol><ol><li>顕微鏡スライド上にドロップ（〜20μl）のVectashieldを置きます。 </li><li>慎重にタングステン針を使用してダウンVectashieldと向きそれらの背側に2つの幼生の標本まで転送する。 </li><li>個別に封入剤から標本をドラッグし、並行してそれぞれの位置を合わせます。 </li><li>試料の両側にカバースリップを配置。鉗子を使用して、後部カバースリップ上の1つの側面を敷設し、そのためのブリッジを形成する前カバースリップにそれを下に下げることによって、最初にトップのサードカバースリップを配置。毛管力は、幼虫の心臓の適切な配置を支援する後方から前方へVectashieldの流れが発生します。 </li><li>マニキュアで、その縁で、場所にすべてのカバースリップを修正し、慎重に20から30μlをVectashieldとカバーグラスの間にスペースを埋める。 </li><li> 4でマニキュアとストアを持つシール° C、または-20℃で長期保存のための</li></ol>代表的な結果正しく実行されると、背脈管のすべてのコンポーネントと関連付けられている組織はそのまま残り、容易に可視化する必要があります。バックグラウンド蛍光は最小限に抑えてください。大人、非常によく腹側縦筋層の汚れのため、実質的な信号（図1および図2）を生成します。根底にある円形の心筋細胞は、しかし、上を覆う腹側層と強烈な信号として生成することではない傾向がある。成人の心臓の前&quot;円錐室&quot;の心筋細胞は収縮物質の相当量が含まれており、その結果、この地域は、心臓の管の残りの部分に最も堅牢な相対として表示されます。幼虫の心臓は大人の収縮心筋細胞（図3）と同様のスパイラル筋原繊維の配置を示しています。 <img src="/files/ftp_upload/1423/1423fig1atmb.jpg" alt="図1a" border="0" /> <img src="/files/ftp_upload/1423/1423fig1btmb.jpg" border="0" alt="図1b" /> 図1：トップ。ミオシン- GFPを発現し、成人キイロショウジョウバエの全心臓のチューブを示す蛍光顕微鏡写真。イメージが余線分スライドモジュールを搭載したツァイスイメージャZ1蛍光顕微鏡で撮影されました。ミオシン- GFPは緑色で示され、アクチンがAlexaFluor ® 594ファロイジン（赤色）とα-アクチニンで染色され、抗α-アクチニン抗体（青）で標識されている。調製手順がよく保存された構造を持つ心臓サンプルの回収を可能に注意してください。 CC =円錐形の容器、AM =翼の筋肉、V =内部バルブ、VLL =腹側縦筋層。ボトム。ちょうど収縮心筋のスパイラル筋原線維の構成を示す腹側長手方向の層の下にある心臓の3番目の腹部のセグメントを介してCCから成人の心臓管の領域。してください<a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg">ここをクリックして</a> 、図1aの拡大版のため、および<a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg">ここで</a> 、図1bの拡大のために<img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="図2" /> 図2：AlexaFluor ® 594ファロイジン（赤色）と抗Pericardin（コラーゲンタイプIV）（緑）抗体で染色された成人の心臓の前方部分の代表的な共焦点スタック。 Pericardinはおそらく腹側筋層の長手方向に指向筋原線維に由来する、腹側表面に沿って心臓に関連付けられています。してください<a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg">ここをクリックして</a>図2の拡大バージョンのために。 <img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="図3" /> 図3：三段階ショウジョウバエの幼虫の適切な心のセグメントA7の蛍光顕微鏡写真。イメージが余線分スライドモジュールを搭載したツァイスイメージャZ1蛍光顕微鏡で撮影されました。アクチンがAlexaFluor ® 594ファロイジン（緑色）とα-アクチニンで標識され、抗α-アクチニン抗体（赤）で染色される。してください<a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg">ここをクリックして</a>図3の拡大バージョンのために。

<ol>
	<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. , (2009).</li><li>Molina, M. R., Cripps, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostia,+the+inflow+tracts+of+the+Drosophila+heart,+develop+from+a+genetically+distinct+subset+of+cardial+cells.">Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.</a> Mech Dev. 1, 51-59 (2001).</li><li>Dulcis, D., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+innervation+of+the+heart+initiates+retrograde+contractions+in+adult+Drosophila+melanogaster.">Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster.</a> J Neurosci. 2, 271-280 (2005).</li><li>Zeitouni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+involved+in+adult+heart+formation+revealed+by+gene+expression+profiling+in+Drosophila.">Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila.</a> PLoS Genet. 10, 1907-1921 (2007).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+change.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological change.</a> J. Morphol. 2, 351-399 (1936).</li><li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The internal anatomy and histology of the imago of Drosophila melanogaster. , (1950).</li><li>Rizki, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulatory+system+and+associated+cells+and+tissues.">The circulatory system and associated cells and tissues.</a> , (1978).</li><li>Cammarato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+transducer+mutations+differentially+affect+motor+function,+myofibril+structure,+and+the+performance+of+skeletal+and+cardiac+muscles.">Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.</a> Mol Biol Cell. 2, 553-562 (2008).</li><li>Taghli-Lamallem, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+deficiency+in+Drosophila+reduces+lifespan+and+causes+a+dilated+cardiomyopathy+phenotype.">Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype.</a> Aging Cell. 2, 237-249 (2008).</li><li>Kelso, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+database+documenting+a+GFP+protein-trap+insertion+screen+in+Drosophila+melanogaster.">a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster.</a> Nucleic Acids Res. 32, D418-D420 (2004).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 7150, 151-156 (2007).</li><li>Mery, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+muscle+LIM+protein,+Mlp84B,+is+essential+for+cardiac+function.">The Drosophila muscle LIM protein, Mlp84B, is essential for cardiac function.</a> J Exp Biol. 211, 15-23 (2008).</li></ol>

ここでは、 キイロショウジョウバエの背の容器と蛍光または共焦点顕微鏡を介してイメージングのための関連する組織を準備し、染色のための有用なプロトコルを提示。私達はによって洗練されたと一般的に幼虫と成体ショウジョウバエの心臓管の in situイメージングでよく解決を可能にする効果的な染色のために私たちの研究室で用いられる手順の簡潔なアカウ?...

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<td>Ethylene glycol-bis (2-amino-ethylether) -N,N,N&#x2019;,N&#x2019;-tetra-acetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>E4378</td>
<td>&nbsp;</td>
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<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
<td>Reagent</td>
<td>Polysciences Inc.</td>
<td>50-00-0</td>
<td>&nbsp;</td>
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<td>Triton-X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
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<td>Alexa Fluor&reg; 594 phalloidin</td>
<td>Reagent</td>
<td>Invitrogen&trade;</td>
<td>A12381</td>
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<td>Vectashield&reg; Mounting Medium for Fluorescence with DAPI</td>
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<td>Vector Laboratories, Inc.</td>
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fluorescent labeling of drosophila heart structures

市販<em&gt;キイロショウジョウバエ</em&gt;背脈管、または心臓は、収縮心筋細胞、心臓の壁の両側に沿って整列心膜細胞、支持翼の筋肉と、成人では、腹側縦筋細胞の層の単層で構成された管状構造である。収縮繊維の家は、筋原線維と細胞外マトリックスの相同成分と相互作用細胞骨格/亜膜性のタンパク質複合体、の密集バンドルを含む筋肉の細胞構築の成分を保存。ここでは、固定するためのプロトコルと解剖の幼虫やセミ無傷の大人の心の中から特定の心筋梗塞の要素の蛍光標識を記述する<em&gt;ショウジョウバエ</em&gt;。具体的には、幼虫の心の中にF -アクチンサルコメアのとα-アクチニンのラベルを示す。さらに、我々はミオシン- GFPは野生型の大人の心で、大人のハエを表現し、α-アクチニンとpericardin、IV型細胞外マトリックスのコラーゲンで、F -アクチンとα-アクチニンの標識行います。特に注意がハンドリングを最小限に抑え、心臓の管と関連した組織の整合性を維持するための機会を最適化する半無傷の大人の心の取り付け戦略に与えられます。これらの製剤は、蛍光と共焦点顕微鏡を介して画像処理に適しています。全体的に、この手順は、強力な遺伝的に扱いやすいモデル系から心臓の構造的な特性を慎重かつ詳細な分析が可能になります。

Watch this Scientific Journal Video about Fluorescent Labeling of Drosophila Heart Structures at JoVE.com

Fluorescent Labeling of Drosophila Heart Structures

ここでは、幼虫および成体から心臓管の様々な要素の蛍光標識するための基本的なプロトコルを記述する<em&gt;キイロショウジョウバエ</em&gt;。これらの標本は、蛍光または共焦点顕微鏡を介して画像処理に適しています。この手法は、強力なモデル生物からの心の機能の詳細な構造解析を可能にします。

の蛍光標識ショウジョウバエハートの構造

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

fluorescent-labeling-of-drosophila-heart-structures

Research

JoVE Journal

Biology

Fluorescent Labeling of Drosophila Heart Structures

13.6K ビュー.  San Diego State University. ここでは、幼虫および成体から心臓管の様々な要素の蛍光標識するための基本的なプロトコルを記述するキイロショウジョウバエ。これらの標本は、蛍光または共焦点顕微鏡を介して画像処理に適しています。この手法は、強力なモデル生物からの心の機能の詳細な構造解析を可能にします。

ビデオ: Fluorescent Labeling of Drosophila Heart Structures

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

生物学

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

ライブの視覚化ショウジョウバエ</em蛍光色素で>グリア - 神経筋接合部

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

の心臓の鼓動の可視化ショウジョウバエ</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

抗体標識用ショウジョウバエ多糸染色体のカボチャの準備

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

COS - 7の蛍光標識は、生細胞イメージングのためにSNAP - Tag融合タンパク質を発現する

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...