我々は、ゼブラフィッシュ畜産で彼の助けのためにBeninio Jomokに感謝。この作品は、NIH HL81753によって運営されている。

1。スライドの準備と加湿チャンバー<ol><li>加湿チャンバーを空にチップボックスのようなボックスから行うことができます。チャンバーと光から試料を保護するためにアルミホイルでカバーの両方を包みます。 </li><li>チャンバー内に、乾燥から心臓サンプルを防ぐことがチャンバ内の湿度を維持するために湿紙タオルの山を置く。スライド用ラックとしてペーパータオルの上に小さなプレートを設定します。 </li><li> 5 × 5 mm程度の寸法と心臓のサンプル用井戸を作るためにimmEdgeのペンを使って顕微鏡スライドの表面に線や円のいずれかを描く。スライドを乾燥させて。 </li><li>加湿チャンバー内のスライドを入れて、各ウェルに50UL新鮮な4％のホルムアルデヒドを追加。 </li></ol> 2。胚の心臓の解剖<ol><li>魚止めのボディの動きまで、約1分間0.4％tricaineを含むE3卵の水7で魚の胚を麻酔がまだ正常な心を持っている鼓動。 </li><li>解剖顕微鏡のステージ上に凹の顕微鏡用スライドを置く。よく凹部に胚を移す。 </li><li>明らかに心を明らかにするために解剖顕微鏡の光の組み合わせを調整します。個人の好みに応じて、暗視野やDICのような偏光状態のいずれかである可能性があります。 </li><li>その後、乾燥、75％エタノールで2つのインスリン注射器を清掃してください。 </li><li>心臓より高い倍率と焦点に解剖顕微鏡を調整します。物理的に頭部に近い卵黄体節境界に1つの針の先端を挿入することによって胚を修正。心臓領域に別の針の近くに挿入し、すぐに心を引き出します。心臓が完全に胚から分離されていない場合は、心臓と卵黄の間に、残りの接続組織を切断。 </li><li>分離された心臓の低倍率と焦点に顕微鏡を調整します。 10μlのpipettemanを使用し、1μlの音量に設定します。どちら描画または触れるためにpipettemanの先端を適用する単離心臓の内でまたは先端の表面に付着するのどちらかになるように心はサンプル、。 </li><li>準備されたスライド上の4％ホルムアルデヒド溶液でピペット先端を浸して溶液中に心を解放するためにピペットピストンを押し下げる。水面の表面張力は、溶液にピペットチップから心を解放するのに役立ちます。各ウェルの3心より小さいを置く。 </li></ol> 3。免疫染色<ol><li>チャンバーを閉じ、穏やかなロッキング運動でシェーカー上に置いた。室温で20分間心臓のサンプルを修正。 </li><li>スライドの裏面に水を拭いてから、交換するバッファのための解剖顕微鏡のステージにスライドを置く。 </li><li>各ウェルに空の領域に10μlのpipettemanから先端を浸し、心臓のサンプルから可能な限り先端を保つ。ソリューションを先端に描画されるときに心が水の流れで移動する可能性がありますので、先端に装着、心臓を避けるために注意してください。変更必要に応じて先端のローカライズ、。 </li><li>組織上に溶液を処分すると心臓のサンプルが容易に気泡の存在下で失われているので、先端に気泡を生成することは避けてください。短い時間のために心臓のサンプルは部分的に乾燥しておくこと心がスライドに付着したままでいることができます。 </li><li>心臓のサンプルをカバーし、5分間ゆっくりと揺動運動の加湿チャンバー内でインキュベートする50μlのPBSTを追加。リンスつのより多くの時間を繰り返します。このステップの後、心臓のサンプルは4℃加湿チャンバーまでの1週間℃で保存することができます。 </li><li>室温で1時間、PBSTで10％のヒツジ血清で心臓のサンプルをブロックする。 </li><li>室温で1時間10％ヒツジ血清/ PBSTで、このようなβ-カテニン抗体の1:200希釈とMEF2特異抗体の1:200希釈などの一次抗体、と心臓のサンプルをインキュベートする。 </li><li>室温でPBST三回（5分。ごと）で心臓のサンプルを洗浄してください。 </li><li>二次でサンプルをインキュベートするPBSTで0.5時間のためのそのようなアレクサタグ付き抗マウスおよび/または抗ウサギ抗体の1:1000希釈のような抗体を、、。 </li><li> PBSTで3回心臓サンプルを洗う（5分。ごと）室温での。 </li></ol> 4。イメージング<ol><li> PBSTを取り外し、各ウェルに10μlの封入剤を加える。溶液が透明になるまで数分間チャンバーを揺らし。 </li><li>封入剤のほとんどを削除し、顕微鏡のカバーガラス（24x50）とのスライドで井戸のすべてをカバーしています。 </li><li>ツァイスAxioplan 2顕微鏡を用いて画像の魚の心臓は、線分（カールツァイス）を装備。 </li></ol> 5。代表的な結果すべてのゼブラフィッシュの心筋細胞の膜と核を明らかに画像の例を図に示します。 1。 MEF2Cとβ-カテニン抗体は52 HPFのゼブラフィッシュの胚から摘出心臓の試料を染色するために一緒に使用されていました。一方MEF2C抗体染色の心筋細胞の核、β-カテニン抗体は、各セル8-10の境界を明らかにする。複合顕微鏡のステージを調整することにより、心臓のサンプルの画像は、外側の曲率と心臓11の内側の曲率の観察を可能にする同じ方向に調整することができます。合計心筋細胞の数、心筋細胞のサイズ、および真円度を測定することができます。 胎児心臓のサルコメア構造を外部にもらす別の例を図に示します。 2。我々は解剖胎児心でF59、ミオシンの抗体を用いて免疫染色を行った。太いフィラメントの紋帯が高い倍率（図2）6で明らかにすることができますながら、全体の胎児心における筋原線維のネットワークを明確に、低倍率で明らかにすることができる。 <img alt="図1" src="/files/ftp_upload/3510/3510fig1.jpg" /> 核とOUTLを表示するMEF2（赤）とβ-カテニン（緑）の図1。免疫染色ゼブラフィッシュ心室の心筋細胞のINES。スケールバー=10μmの<img alt="図2" src="/files/ftp_upload/3510/3510fig2.jpg" /> どちらかの低倍率（）または高倍率（B）でゼブラフィッシュ脳室に太いフィラメントを表示するミオシンの図2。Immnostaining。スケールバー=10μmの

<ol>
	<li>Shin, J. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Zebrafish+to+human:+modular+medical+models.">From Zebrafish to human: modular medical models.</a> Annu. Rev. Genomics Hum. Genet. 3, 311-340 (2002).</li><li>Beis, D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology:+following+the+zebrafish+trend.">In vivo cell biology: following the zebrafish trend.</a> Trends. Cell. Biol. 16, 105-112 (2006).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin. Cell. Dev. Biol. 18, 27-35 (2007).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Dahme, T., Katus, H. A., Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+the+genetic+basis+of+cardiovascular+disease.">Fishing for the genetic basis of cardiovascular disease.</a> Dis. Model Mech. 2, 18-22 (2009).</li><li>Huang, W., Zhang, R., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+zebrafish+heart:+A+functional+study+of+tnnt2.">Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.</a> Dev. Biol. 331, 237-249 (2009).</li><li>Westerfield, M. <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 Zebrafish Book. , (2007).</li><li>Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle:+the+regulation+of+myogenesis.">Muscle: the regulation of myogenesis.</a> Curr. Opin. Genet. Dev. 4, 745-751 (1994).</li><li>Wheelock, M. J., Knudsen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+and+associated+proteins.">Cadherins and associated proteins.</a> Vivo. 5, 505-513 (1991).</li><li>Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy+involves+both+myocyte+hypertrophy+and+hyperplasia+in+anemic+zebrafish.">Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.</a> PLoS One. 4, e6596-e6596 (2009).</li><li>Auman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modulation+of+cardiac+form+through+regionally+confined+cell+shape+changes.">Functional modulation of cardiac form through regionally confined cell shape changes.</a> PLoS Biol. 5, e53-e53 (2007).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+zebrafish+essential+and+regulatory+myosin+light+chains+reduces+cardiac+function+through+distinct+mechanisms.">Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms.</a> Cardiovasc. Res. 79, 97-108 (2008).</li></ol>

利害の衝突は宣言されません。

古典的な全体のマウント免疫染色の方法に比べ、本手法は次のような利点があります。最初に、はるかに強い蛍光シグナルが一貫して改善された浸透のために取得することができます。全体のマウント免疫染色法では、心臓を取り巻く濃い皮膚組織は大幅に全身に高グラウンドで、その結果、多くの抗体の浸透を減少させた。この問題は、3日後に受精（DPF）より古い胚には特に厳しいです。...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> tricaine </td><td>研究の有機物</td><td> 3007T </td></tr><tr><td>ホルムアルデヒド</td><td> Polysciences </td><td> 04018 </td></tr><tr><td>インスリン注射器</td><td>ベクトンディッキンソン</td><td> 329461 </td></tr><tr><td>トリトンX - 100 </td><td>シグマ</td><td> T8532 </td></tr><tr><td>抗β-カテニン抗体</td><td>シグマ</td><td> C7207 </td></tr><tr><td>抗MEF2C抗体</td><td>サンタクルスバイオテクノロジー</td><td> SC313 </td></tr><tr><td> F59 </td><td> ZFIN </td><td></td></tr><tr><td>抗α-アクチニン抗体</td><td>シグマ</td><td> A7811 </td></tr><tr><td>抗ILK抗体</td><td>細胞シグナリング</td><td> ＃3862 </td></tr><tr><td>のAlexa Fluor ® 568ヤギ抗ウサギIgG抗体</<html> TD&gt; <td>インビトロジェン</td><td> A11011 </td></tr><tr><td>のAlexa Fluor ® 488ヤギ抗マウスIgG1 </td><td>インビトロジェン</td><td> A21121 </td></tr><tr><td>蛍光用封入剤</td><td>ベクトル</td><td> H - 1200 </td></tr><tr><td> ImmEdgeペン</td><td>ベクトル</td><td> H - 4000 </td></tr><tr><td>ポリ- L -リシンコートしたスライド</td><td>電子顕微鏡学</td><td> 63410〜01 </td></tr><tr><td>顕微鏡のカバーガラス</td><td>フィッシャー</td><td> 12から543 - D </td></tr><tr><td>凹んだ顕微鏡スライド</td><td>フィッシャー</td><td> 7-1305-8 </td></tr><tr><td>解剖顕微鏡</td><td>ライカMZ95 </td><td></td></tr><tr><td>化合物の顕微鏡</td><td>ツァイス</td><td> Axioplan2 </td></tr><tr><td>線分</td><td>ツァイス</td><td></td></tr></tbody></table> PBST： 1 × PBS 0.5％トリトンX - 100 ENT&quot;&gt; 25X Tricaine： 400 mgのTricaine 97.9 mLののddH 2 O 2.1 mLの（1MトリスPH9） pHを7.0に調整する

immunostaining of dissected zebrafish embryonic heart

ゼブラフィッシュの胚は、心臓の開発とその有利な発生学と遺伝学1,2のために人間の心の病気を研究するためのin vivoで脊椎動物のモデルで人気となる。約100-200胚は、成魚の単一ペアから毎週容易に入手可能である。 EX子宮を開発透明な胚は、心臓の欠陥3を評価するために最適です。任意の遺伝子の発現は、モルホリノ技術またはRNAの注射4を介して操作することができます。また、前方に遺伝子スクリーニングは、すでに心臓発生5の様々な観点に影響を与える変異体のリストを生成している。 全体のマウント免疫染色では、特定の組織6に標的タンパク質の発現パターンを明らかにするためには、この動物モデルにおいて重要なテクニックです。しかし、携帯電話または細胞内構造を明らかにすることができる高解像度の画像は、主に物理的なlocatに、難しい状態が続いている心と抗体のほとんど通過のイオン。 ここで、我々は最初の心臓を解剖し、顕微鏡スライドの表面に染色処理を行うことにより、これらのボトルネックに対処する方法を提示する。小さな心臓サンプルの損失を防ぐために、ソリューションの取り扱いを容易にするために、我々はimmEdgeのペンによって描画される顕微鏡の表面をスライド上に円の中で心臓のサンプルを制限。染色後、蛍光シグナルを直接複合顕微鏡で観察することができます。 胚魚の心臓は、わずか数細胞層で構成されて以来、私たちの新しいメソッドは、抗体の浸透を大幅に向上します。無傷の心からの高品質の画像が2日から6日に歳のゼブラフィッシュ胚のために多くの削減行列の時間内に得ることができます。我々の方法は、潜在的にゼブラフィッシュや他の小動物のいずれかから切除した他の器官を染色するために拡張することができます。

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

ゼブラフィッシュ胚の心臓の免疫染色を実施する迅速な方法が説明されています。全体のマウント免疫染色の手法に比べ、この方法は劇的に非常に低下処理時間内に心臓の細胞/細胞内構造を明らかに高解像度の画像を得ることができる抗体の浸透を、増加させる。

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.6K ビュー.  Mayo Clinic College of Medicine. ゼブラフィッシュ胚の心臓の免疫染色を実施する迅速な方法が説明されています。全体のマウント免疫染色の手法に比べ、この方法は劇的に非常に低下処理時間内に心臓の細胞/細胞内構造を明らかに高解像度の画像を得ることができる抗体の浸透を、増加させる。

ビデオ: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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 ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

ゼブラフィッシュ胚眼の組織の顕微解剖

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

胚肢の皮膚の血管系のためのホールマウント免疫組織化学的解析：胚における血管分岐形態形成を研究するモデルシステム

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

解離調製物から胚および幼虫のゼブラフィッシュ骨格筋線維の分析

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...