著者は、技術の発展に原稿の生産に貢献しダウリングラボのメンバー（アーロンReifler、トレント·ウォー、アンジェラバスタ、ウィリアム·テルファー）に感謝したい。この作品は、トーブマン研究所、ミシガン大学の小児科、および筋ジストロフィー協会（JJD MDA186999）と国立衛生研究所（JJD 1K08AR054835）からの補助金の一部はによって資金を供給された。

1。ポリ-L-リジンコートしたカバーガラスの製造（時間：1時間） コー​​ティングしたカバーは、急速な筋線維の沈降との密着性を可能にします。これは、筋線維アイソレーション（以下、工程2）の解離工程中に行ってもよい。 <ol><li>カットして60ミリメートルペトリ皿（どのブランド）の底にパラフィルムを配置します。 </li><li> 60ミリメートルの組織培養皿にパラフィルム上の顕微鏡カバーガラススリップ（直径12mm）を配置し、24ウェルプレートの単一のウェルに個別に配置します。 </li></ol>注1：これらの小さな丸いカバースリップは、筋線維数を集中し、過剰な抗体の使用量を減らすのに役立つ。 注2：他のサイズのカバーグラスは動作しますが、使用した試薬および胚のボリュームは、それに応じて調整する必要があります。 <ol start="3"><li>ピペットシャーレ内の各カバーガラス上にポリ-L-リジン溶液（0.01％）の50〜200μL。 </li><li>カバースリップは、少なくとも1のHO座ってできるように37℃でのUR長いインキュベーションは否定的な結果には影響しません。 </li><li>カバーガラスからポリ-L-リジン溶液を除去し、100μlの1X PBS最小で二回洗う。 </li><li>カバーグラスを乾燥させる。 </li><li>カバースリップは今筋線維の準備ができている。 </li></ol>注3：無菌性を保証するために、コーティングはフード内でかつオートクレーブカバースリップ上で行うことができる。 注4：底にパラフィルムで60ミリメートルペトリ皿にしてください。このセットアップでは、筋線維のメッキと免疫標識（後の手順）中に使用されます。 代替案1：代わりに、使用直前にコーティングカバースリップを、コーティングされたカバースリップのストックを使用することができる。 2ミリリットルのポリ-L-リジンの最小値を含む60ミリメートルのペトリ皿は、多数のカバースリップを含有する4℃で保存することができる。この代替では筋線維のためのカバースリップを処理するためにステップ1.5から開始します。 代替2：私たちも、ポリ-L-オルニチンでのコートカバースリップ。 T彼は、より労働集約的であるが、ポリ-L-オルニチンでコーティングしたカバーガラスをUV処理することができるので、長期培養に有用である。 UV処理と慎重な無菌テクニックと一緒に暮らすの筋線維は、一般的に4-7日の培養で維持することができる。 2。筋線維のゼブラフィッシュ胚とメッキ（：1から3時間の時間）の解離注：標準のプロトコルは3 DPF（日後受精）の胚に最善適用されます。 <ol><li>標準1.5ミリリットル遠心管にゼブラフィッシュ胚を移し、可能な限り余分な魚の水を除去する。典型的には、より少ないけれどもチューブ当たり10〜20の胚を使用することができる。以上の20〜25の胚を使用すると、多くの場合、過剰な濃度の製剤が得られる。 <ol><li> ＃5鉗子を使用して手動で、解離前絨毛膜を除去します。あるいは、化学的絨毛膜の除去は、10〜15分間プロナーゼ処理によって達成される。一般的に、絨毛膜の前の除去にのみ必要とされるときMUTの事前確認アリ表現型または形態が必要とされる、または段階を使用するときに胚を自然に絨毛膜から孵化される場合。 </li></ol></li><li>胚を含むチューブに、CO 2の独立したメディアの900を添加する。 </li><li> 100コラゲナーゼタイプIIのμL、最終濃度3.125 mg / mlの解離を開始します（31.25ミリグラム/ 1X PBSで希釈したミリリットルの在庫コラゲナーゼ溶液）を追加します。コラゲナーゼIVはまた、解離のために使用することができる。 </li><li>オービタルシェーカー上で胚を回転させ、チュレーション用P1000ピペットマンを使用して、室温で30分ごとに粉砕する。 </li></ol>注2：慎重に胚の解離を監視し、プロトコル障害の最も一般的な理由である以上、または解離の下（特に上で）。消化のための時間は粉砕、チューブあたり胚の数、および胚の時代の強さに応じて異なります。また、骨格筋の変異体からの胚（野生型に比較して）以下であることが多い。 ノート3：胚歳あたりの平均消化時間= 1時間1 DPF、2 DPF = 1.5時間、3 DPF = 2時間、および= 2-2.5時間4 DPF。 <ol start="5"><li>何胚全体が表示されていない、まだ固体部分がまだ表示されているとき、消化を停止する- これは過剰消化を防ぐために不可欠です。 </li><li>ペレット細胞に3〜5分間0.8から2.3 XGで解離胚と遠心管。 </li><li>ペレット化した細胞からの上清を除去し、CO 2の独立したメディアで2回洗う。 </li><li>細胞を再懸濁するため、新鮮なCO 2の独立したメディアを追加します。 1mlの典型的に10〜20胚のプレップのために使用される。ボリュームは、胚番号に基づいてスケーリングすることができる。 </li><li> P1000ピペットを用いて、70μmのフィルターを介して胚サスペンションを渡します。これは準備から不要な破片を除去するのに役立ちます。 </li></ol>注4：胚の懸濁液は、さらに不要な破片を除去するために40μmのフィルターを通して再度濾過することができる。二重ろ過から、回復がapproximです1ミリリットルの出発体積からately 800μL。 <ol start="10"><li>各ポリ-L-リシン被覆カバーガラスに筋線維懸濁液の約50〜100μLを加える。 </li></ol>注5：底にパラフィルムでペトリ皿のステップ2.9を実行して、先に調製した。パラフィルムをコートしたカバーガラスをオフに実行しているから、筋線維のサスペンションを保持します。蒸発を防ぐためにカバーされたペトリ皿にしてください。 <ol start="11"><li>筋線維は、室温でコーティングしたカバーガラス上に約1時間を決済することができます。 </li></ol>注6：筋線維は、5〜10分以内に安定し始めます。しかしながら、良好な筋線維結合のために、1時間（1〜2時間）の最小を推奨します。筋線維が長いために解決できるようにすると、準備に害を与えない。 （一晩を含む）より長いインキュベーションのために、抗生物質を培地に添加することができる。抗生物質や、無菌技術を用いて生きた培養は通常、1〜2日間維持することができる。 <ol start="12"><li>ライブMyofibersこの時点で観察することができる。胚2 DPFの筋繊維、後に横紋と細長い細胞（ 図1）として認識されます。この時点では、筋線維現在ライブ解析用または免疫標識の準備ができている。 </li></ol>注7：胚DPF 1からの骨格筋は、細長い明確横紋繊維プレートはありません。その代わりに、大規模なmyoballsが表示されます。また、ペレット化相ポスト解離（ステップ2.6）中に、1のdpf myoballsペレットを達成するために、5000×gで8分間遠心分離し、最低される必要がある。この段階で胚の解析のために、骨格筋23で特異的にEGFPを発現するトランスジェニックラインを使用することをお勧めします。これは、他のソース対筋起源からの細胞の同定を可能にする。 3。解離ゼブラフィッシュ筋線維の蛍光免疫染色（時間：約1日） 3.1。免疫標識<ol><li>の一部を除去コー​​ティングされたカバーガラスに付着した筋線維からメディア</li><li> 4％パラホルムアルデヒドやメタノールを用いて細胞を固定します。 PFAの場合は、メディアの容量½削除して、PFA、同量と交換してください。 、総ボリュームを削除し、PFAの50〜100μlで交換し、さらに10分間固定した後、室温で10分間固定してください。アルコール固定のために、氷冷メタノールまたはアセトンは、それぞれ、10分又は5分間、4℃で適用することができる。 </li><li>筋線維を少なくとも1×PBSまたは1x PBSで3-5X +0.1％のTweenを洗う。平均洗浄量は、カバースリップあたり50〜100μLです。 </li><li>筋線維を解決（作業ストック）をブロックに追加し、室温で20〜60分間インキュベートする。ブロッキング溶液は、一次および二次抗体によって異なります。実験室で使用される2つの最も一般的なものは、（1）1×PBS、2 mg / mlのBSA、1％ヒツジ血清、0.25％トリトンX-100を最終的なものであり、（2）0.2％トリトンX-100、2 mg / mlのである（0.2 ％のBSA）および5％ヒツジ/ヤギ血清。 </li><li>ブロッキング溶液外し、Bのいずれかで希釈した一次抗体を追加溶液又はPBSをロックする。 4℃で一晩筋線維をインキュベートパラフィルムのいずれかにいることを確認し、または抗体でコーティングされた筋線維にロードされたカバースリップを含むペトリ皿をサランラップでラップします。湿らせたペーパータオルの小片を、加湿チャンバを作成するために添加することができる。 </li><li>カバースリップからの一次抗体を除去し、PBSまたはブロッキング溶液で5分間カバーグラスを3-5Xを洗う。 </li><li> 1X PBSまたはRTで1〜2時間ブロッキング溶液中に希釈した適切な濃度で二次抗体を加える。 </li><li>二次抗体を除去し、室温で5分間ずつ筋繊維PBS中の3-5Xを洗う。 </li></ol> 3.2。マウントカバースリップ<ol start="9"><li>顕微鏡スライドに退色防止剤の1〜2滴を適用します。 </li><li>慎重にピックアップコーティングされたとimmunolabledカバーガラスをピンセットと場所（筋線維ダウン）を使用して、顕微鏡スライド上の退色防止剤にカバースリップを。 </li></ol>注1：Cの向きに注意oatedカバーガラスは非常に重要です。 <ol start="11"><li>硬い固体表面上に1〜2キムワイプを置く。すぐに退色防止剤に載っコートしたカバーガラスとスライドを反転させ、キムワイプ上（下カバースリップ）に置きます。 </li><li>過剰退色を削除し、スライドとカバーガラスの間に緊密なシールを形成するために、顕微鏡スライドの隅に光の圧力を加える</li><li>退色はRTで5〜10分間乾燥さ残りを許可し、筋線維は、画像（ 図2AおよびB）または4℃で保存する準備が整いました</li></ol> 4。 GCaMP3を使用した生細胞カルシウムイメージング生きた細胞実験の前（ステップ2.10以下）に固定筋線維を行うことができる。次のプロトコルはGCaMP3 24、骨格筋の特定のゼブラフィッシュのα-アクチンプロモーター（pSKM）23で表さ遺伝的にコード化されたカルシウム指示薬を発現する筋線維のライブイメージングを説明します。別の方法として、この技術は、容易にこのようなフラ-2カルシウムインジケーター色素を使用するように適合させることができる。 <ol><li> pSKMで胚を注入：GCaMP3は1〜2細胞期の構築</li><li> 3 DPFで、幼虫を収集し、セクション1と2で説明したように筋線維を準備。筋線維は、1時間の最低付着することができます。この技術については、24ウェル皿にカバーガラスを製造することが必要である。 </li><li>慎重に余分なメディアを取り出して、室温で新鮮なCO 2の独立したメディアの300μlを加える。 </li><li>倒立顕微鏡で細胞を観察します。 GCaMP3正筋線維は、緑色の蛍光の下で表示されるはずです。 </li><li>必要に応じて、記録するためのカメラをセットアップします。 </li><li> CO 2の独立したメディアが30mmのカフェイン溶液を調製する。細胞を刺激するために、ゆっくりと拡大してウェルにカフェイン溶液を300μlをピペットで。 5月10日秒以内、GCaMP正筋線維は、蛍光の急激な増加（ 図3）とカフェインに反応する必要があります。 MyofibERSは、契約があります。注目すべきは、カフェインは、筋小胞体店25からのカルシウム放出を誘導する。例えば、26のKCl及びリアノジン4などの他の薬剤は、誘導性カルシウム放出を研究するために使用することができる。 </li></ol>

<ol>
	<li>Dulhunty, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation-contraction+coupling+from+the+1950s+into+the+new+millennium.">Excitation-contraction coupling from the 1950s into the new millennium.</a> Clin. Exp. Pharmacol. Physiol. 33, 763-772 (2006).</li><li>Bannister, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+the+myoplasmic+gap:+recent+developments+in+skeletal+muscle+excitation-contraction+coupling.">Bridging the myoplasmic gap: recent developments in skeletal muscle excitation-contraction coupling.</a> J. Muscle Res. Cell Motil. 28, 275-283 (2007).</li><li>Ohno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+defects+and+disorders+at+the+neuromuscular+junction.">Genetic defects and disorders at the neuromuscular junction.</a> Brain Nerve. 63, 669-678 (2011).</li><li>Lanner, J. T., Georgiou, D. K., Joshi, A. D., Hamilton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryanodine+receptors:+structure,+expression,+molecular+details,+and+function+in+calcium+release.">Ryanodine receptors: structure, expression, molecular details, and function in calcium release.</a> Cold Spring Harb. Perspect. Biol. 2, (2010).</li><li>Dolphin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+diversity:+multiple+roles+of+calcium+channel+subunits.">Calcium channel diversity: multiple roles of calcium channel subunits.</a> Curr. Opin. Neurobiol. 19, 237-244 (2009).</li><li>Sparrow, J. C., Schock, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+initial+steps+of+myofibril+assembly:+integrins+pave+the+way.">The initial steps of myofibril assembly: integrins pave the way.</a> Nat. Rev. Mol. Cell Biol. 10, 293-298 (2009).</li><li>Lapidos, K. A., Kakkar, R., McNally, E. 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+dystrophin+glycoprotein+complex:+signaling+strength+and+integrity+for+the+sarcolemma.">The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma.</a> Circ. Res. 94, 1023-1031 (2004).</li><li>Bassett, D., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+zebrafish+model+of+muscular+dystrophy.">Identification of a zebrafish model of muscular dystrophy.</a> Clin. Exp. Pharmacol. Physiol. 31, 537-540 (2004).</li><li>Barresi, R., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystroglycan:+from+biosynthesis+to+pathogenesis+of+human+disease.">Dystroglycan: from biosynthesis to pathogenesis of human disease.</a> J. Cell Sci. 119, 199-207 (2006).</li><li>Emery, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+frequencies+of+inherited+neuromuscular+diseases-a+world+survey.">Population frequencies of inherited neuromuscular diseases-a world survey.</a> Neuromuscul. Disord. 1, 19-29 (1991).</li><li>Nance, J. R., Dowling, J. J., Gibbs, E. M., Bonnemann, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+myopathies:+an+update.">Congenital myopathies: an update.</a> Curr. Neurol. Neurosci. Rep. 12, 165-174 (2012).</li><li>Bassett, D. I., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+muscular+dystrophy+and+congenital+myopathy.">The zebrafish as a model for muscular dystrophy and congenital myopathy.</a> Hum. Mol. Genet. 12, 265-270 (2003).</li><li>Dooley, K., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+human+disease.">Zebrafish: a model system for the study of human disease.</a> Curr. Opin. Genet. Dev. 10, 252-256 (2000).</li><li>Dowling, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+myotubularin+function+results+in+T-tubule+disorganization+in+zebrafish+and+human+myotubular+myopathy.">Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.</a> PLoS Genet. 5, (2009).</li><li>Dowling, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+successful+antioxidant+treatment+in+models+of+RYR1-related+myopathy.">Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy.</a> Brain. 135, 1115-1127 (2012).</li><li>Kawahara, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+a+zebrafish+model+of+Duchenne+muscular+dystrophy.">Drug screening in a zebrafish model of Duchenne muscular dystrophy.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 5331-5336 (2011).</li><li>Detrich, H. W., Westerfield, M., M, L. I., Zon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+Zebrafish+system.">Overview of the Zebrafish system.</a> Methods Cell Biol. 59, 3-10 (1999).</li><li>Nixon, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+caveolin-associated+muscle+disease;+caveolin-3+is+required+for+myofibril+organization+and+muscle+cell+patterning.">Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning.</a> Hum. Mol. Genet. 14, 1727-1743 (2005).</li><li>Schredelseker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+1a+subunit+is+essential+for+the+assembly+of+dihydropyridine-receptor+arrays+in+skeletal+muscle.">The beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17219-17224 (2005).</li><li>Telfer, W. R., Nelson, D. D., Waugh, T., Brooks, S. V., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=neb:+a+zebrafish+model+of+nemaline+myopathy+due+to+nebulin+mutation.">neb: a zebrafish model of nemaline myopathy due to nebulin mutation.</a> Dis. Model Mech. 5, 389-396 (2012).</li><li>Dowling, J. J., Low, S. E., Busta, A. S., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+MTMR14+is+required+for+excitation-contraction+coupling,+developmental+motor+function+and+the+regulation+of+autophagy.">Zebrafish MTMR14 is required for excitation-contraction coupling, developmental motor function and the regulation of autophagy.</a> Hum. Mol. Genet. 19, 2668-2681 (2010).</li><li>Schredelseker, J., Dayal, A., Schwerte, T., Franzini-Armstrong, C., Grabner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proper+restoration+of+excitation-contraction+coupling+in+the+dihydropyridine+receptor+beta1-null+zebrafish+relaxed+is+an+exclusive+function+of+the+beta1a+subunit.">Proper restoration of excitation-contraction coupling in the dihydropyridine receptor beta1-null zebrafish relaxed is an exclusive function of the beta1a subunit.</a> J. Biol. Chem. 284, 1242-1251 (2009).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Dev. Biol. 192, 289-299 (1997).</li><li>Tian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-sense+mutations+in+the+dihydropyridine+receptor+beta1+gene,+CACNB1,+paralyze+zebrafish+relaxed+mutants.">Non-sense mutations in the dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrafish relaxed mutants.</a> Cell Calcium. 39, 227-236 (2006).</li><li>Dora, K. A., Doyle, M. P., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+calcium+in+smooth+muscle+causes+endothelial+cell+generation+of+NO+in+arterioles.">Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 6529-6534 (1997).</li><li>Hirata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=accordion,+a+zebrafish+behavioral+mutant,+has+a+muscle+relaxation+defect+due+to+a+mutation+in+the+ATPase+Ca2++pump+SERCA1.">accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1.</a> Development. 131, 5457-5468 (2004).</li><li>van Eeden, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+somite+formation+and+patterning+in+the+zebrafish,+Danio+rerio.">Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.</a> Development. 123, 153-164 (1996).</li><li>Gupta, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+dag1+mutant:+a+novel+genetic+model+for+dystroglycanopathies.">The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies.</a> Hum. Mol. Genet. 20, 1712-1725 (2011).</li><li>Leuranguer, V., Papadopoulos, S., Beam, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+calcium+channel+beta1a+subunits+in+triad+junctions+in+skeletal+muscle.">Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle.</a> J. Biol. Chem. 281, 3521-3527 (2006).</li><li>Capote, J., Bolanos, P., Schuhmeier, R. P., Melzer, W., Caputo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transients+in+developing+mouse+skeletal+muscle+fibres.">Calcium transients in developing mouse skeletal muscle fibres.</a> J. Physiol. 564, 451-464 (2005).</li><li>Adams, B. A., Tanabe, T., Mikami, A., Numa, S., Beam, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramembrane+charge+movement+restored+in+dysgenic+skeletal+muscle+by+injection+of+dihydropyridine+receptor+cDNAs.">Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs.</a> Nature. 346, (1990).</li><li>Sakowski, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+study+motor+neurons+from+zebrafish+embryos+and+larvae+in+culture.">A novel approach to study motor neurons from zebrafish embryos and larvae in culture.</a> J. Neurosci. Methods. 205, 277-282 (2012).</li><li>Nakano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+GPI-anchored+proteins+is+essential+for+surface+expression+of+sodium+channels+in+zebrafish+Rohon-Beard+neurons+to+respond+to+mechanosensory+stimulation.">Biogenesis of GPI-anchored proteins is essential for surface expression of sodium channels in zebrafish Rohon-Beard neurons to respond to mechanosensory stimulation.</a> Development. 137, 1689-1698 (2010).</li><li>Davidson, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+deletion+of+lysine+7+expands+the+clinical,+histopathological+and+genetic+spectrum+of+TPM2-related+myopathies.">Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.</a> Brain. 136, 508-521 (2013).</li><li>Schredelseker, J., Shrivastav, M., Dayal, A., Grabner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Ca2+-conducting+Ca2++channels+in+fish+skeletal+muscle+excitation-contraction+coupling.">Non-Ca2+-conducting Ca2+ channels in fish skeletal muscle excitation-contraction coupling.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5658-5663 (2010).</li></ol>

著者らは、競合する利害を宣言していません。

ゼブラフィッシュは、 生体内で 25,27,28 での筋肉の発達や機能を研究するための強力な脊椎動物のモデルシステムである。彼らはまた、人間の筋肉の病気14,15,20,29をモデル化するための貴重な資産として浮上している。長足の進歩は、筋機能および筋肉疾患の研究のためにゼブラフィッシュを使用して、アプリケーションを進めるためにとられているが、遺伝的?...

骨格筋は運動性のために必要な収縮力を発生させるための責任高度に特殊化組織である。収縮は、細胞内貯蔵1,2からのカルシウム放出に電気信号に変換興奮収縮（EC）のカップリングと呼ばれるプロセスを介して開始される。細胞内カルシウム放出は、力を短縮し、生成するサルコメアをアクティブにします。神経筋接合部の伝達3、ECカップリング4,5、およびアクチン-ミオシン依存性収縮6の媒介に関与する分子機構の多くは、特定のコンポーネントには、熱心な研究の継続的な主題であり続ける。また、収縮7,8および筋線維と細胞外マトリックスの7,9の間のシグナリング、その仲介の間に筋肉の膜を安定させるタンパク質が同定されており、非常に詳細に研究した。 筋肉構造aの重要な多数の遺伝子における変異番目の関数は、（ヒト骨格筋疾患の原因として同定されている<a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ）。臨床的および組織病 ​​理学的特徴に基づく骨格筋障害や筋ジストロフィーなど大きく分けて、これらの疾患は、筋力低下、生涯障害、早期死亡率10,11に関連している。ゼブラフィッシュは、モデリングおよびヒト骨格筋疾患8,12,13を研究するための優れたシステムであることが証明されている。それは、新たな遺伝子変異8を検証する疾患病態生理学14,15の新たな側面を定義し、新たな治療アプローチ15,16を同定するために使用されている。ヒトの筋肉疾患を研究するためのゼブラフィッシュのパワーが仔多数の筋構造および機能の急速な発展は、ゼブラフィッシュ胚の光学的透明度、及び現像シマウマの遺伝的および薬理学的操作の容易さに関する魚17。 私たちは、他の12,18,19は最近開発ゼブラフィッシュからの筋線維の迅速かつ効率的に分離するための簡単な技術を開発した。この方法論は、全胚分析によって提供できるよりもより詳細に筋線維の検査を可能にした。技術は、タンパク質の細胞内局在20の特徴付けだけでなく、新たに開発された疾患モデル21における検証研究の一環として、重要な組織病 ​​理学的特徴の識別に利用されてきた。さらに、孤立した筋線維は、さらにライブイメージング用および電気生理学的研究22、筋肉機能の重要な側面の問い合わせを可能に技術を使用することができます。その後の分析的実験の2つの例と一緒に筋線維の分離のための特定のプロトコルは、この原稿の残りの部分で詳しく説明している。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Invitrogen Gibco</td>
			<td>70011</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Independent medium</td>
			<td>Invitrogen Gibco</td>
			<td>18045</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington Biochemical</td>
			<td>LS004186</td>
			<td>Lot 41H12764</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington Biochemical</td>
			<td>L5004188</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fischerbrand</td>
			<td>12-545-82 12CIR-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#945;-Actinin antibody</td>
			<td>Sigma</td>
			<td>A5044</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RYR antibody</td>
			<td>Abcam</td>
			<td>34C</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor antibody</td>
			<td>Invitrogen</td>
			<td>A-21425</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Fischerbrand</td>
			<td>0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Fischerbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma</td>
			<td>C0750</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations

ゼブラフィッシュは、骨格筋機能を探索するため、およびヒト筋疾患を研究するための貴重なモデル系であることが証明された。ゼブラフィッシュにおける骨格筋のin vivo解析によって提供される多くの利点にもかかわらず、特に全体の胚では、筋肉の機能を担う複雑かつ微細構造化タンパク質環境を可視化する、問題となる可能性がある。この障害は、ゼブラフィッシュの骨格筋（60ミクロン）のサイズが小さく、筋節のさらに小さいサイズに起因しています。ここでは、説明し、ゼブラフィッシュの胚および幼生から骨格筋線維を分離するための簡単​​かつ迅速な方法を示しています。また、筋肉の構造および機能を分析するための便利なポストの調製手法を例示するプロトコルを含む。具体的には、詳細骨格筋タンパク質のその後の免疫細胞化学的局在化および生細胞カルシウムイメージングを介して刺激されたカルシウム放出の定性分析。全体として、このビデオ記事では、ゼブラフィッシュの骨格筋線維の単離および特徴、筋肉の構造と機能の無数のその後の研究のための導管を提供して技術が素直で効率的な方法を提供する。

筋線維の蛍光免疫標識（ 図2） 筋線維から予想される蛍光標識パターンを示す画像が正常な分離とめっき後に免疫染色。筋線維は抗リアノジン受容体（1:100）（ 図2A）または抗α-アクチニン（1:100）（ 図2B）抗体のいずれかで標識し、それぞれトライアドとZ帯の免疫染色を明らかにしてきた。使用した二次抗体は、アレクサフ...

Watch this Scientific Journal Video about Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations at JoVE.com

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

ゼブラフィッシュは、骨格筋のヒト障害をモデル化するための新たなシステムである。私たちは、胚および幼虫のゼブラフィッシュから骨格筋筋線維を隔離するために迅速かつ効率的な方法が記載されている。この方法は、単一の骨格筋線維の形態、タンパク質の細胞内局在、および筋生理学の研究に適した高密度の線維調製物が得られる。

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 ...

analysis-embryonic-larval-zebrafish-skeletal-myofibers-from

Research

JoVE Journal

Biology

11.5K ビュー.  University of Michigan. ゼブラフィッシュは、骨格筋のヒト障害をモデル化するための新たなシステムである。私たちは、胚および幼虫のゼブラフィッシュから骨格筋筋線維を隔離するために迅速かつ効率的な方法が記載されている。この方法は、単一の骨格筋線維の形態、タンパク質の細胞内局在、および筋生理学の研究に適した高密度の線維調製物が得られる。 

ビデオ: Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

遺伝子発現解析のための胚ゼブラフィッシュとcDNA合成からのRNA分離

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

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 ...

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

イメージングおよび胚ゼブラフィッシュにおける脳血管構造物の3次元再構成

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.