我々は、技術的なヘルプやアドバイスを貰えるボブ（アンドールテクノロジー）に感謝。 SA-S。ドイツ学術振興協会（DFG）のハイゼンベルク交わりによってサポートされています。この作品は、助成SE2016/7-1をDFGによってサポートされていました。

1。単一細胞を標識 β-アクチンプロモーターの制御下でGFPをコードするプラスミドまたは他のGFP融合タンパク質と1細胞期胚を注入します。開発中に、GFPはその後モザイク様式で発現されています。 、β-アクチンプロモーターの制御下にα-アクチニン-GFP融合タンパク質を置きます：ここでは、トランスジェニック構築TG [α-アクチニン-GFPのβ-アクチン ]を使用していました。 2。胚の埋め込み<ol class="lalpha"><li>ゼブラフィッシュ胚を採取し、28 HPF（ステージングが12による）まで、通常の条件下で維持します。 </li><li>鉗子（デュモン＃5）と実体顕微鏡下で胚Dechorionate。 </li><li>ワセリンリング付き顕微鏡スライドを準備します。 </li><li> E3の培地で1％低融点アガロース/ 10％tricaine溶液を調製：液体および均質アガロース溶液を得るために10ミリリットルのE3溶液中で沸騰100mgのアガロース、アリコートとストアさらに使用するために-20℃で未使用のアガロース溶液。すぐに使用するときは、ゆでたソリューションが冷えるとmaxで維持することができます。 39℃ 10パーセントtricaineの最終濃度を得るためにtricaine原液を追加します。 tricaineとアガロースの両方が胚の動きを防ぐために必要です。 </li><li>胚が側に自然に敷設して、ワセリンリング内の1％低融点アガロース/ 10％tricaine / E3の媒体における単一胚を埋め込む。優しくだけの場所で胚を維持し、凸を避けるためにカバーガラスで覆う光を壊してしまう表面。理想的には、ターゲットとなる胚筋組織は、カバーガラスに触れるべき。 </li></ol> 3。胚レーザー負傷<ol class="lalpha"><li> micropointレーザーと顕微鏡を準備します。 <ol class="lroman"><li>顕微鏡と組み合わせて設定し、レーザーを使用する方法の詳細についてはmicropointレーザーマニュアルを参照してください。 </li><li>正しいを選択435 nmの波長のレーザ光を生成するための染料。 </li><li>減衰スライダを使って、レーザーパワーを調整します。レーザーパワーは、単一パルス（これをテストするサンプルのカバーガラス上などフォーカス）でガラス表面を壊すに十分な強さがあります。これは、細胞アブレーションに必要なレーザーパワーである。 </li></ol></li><li>顕微鏡の接眼レンズに搭載されたレチクルを用いて関心領域またはセルにフォーカス。 20倍の対物レンズを（40Xより正確なダメージが可能になりますが、多くの場合、作動距離がこのレンズのために十分ではありません）を使用します。 </li></ol> 注1：レーザーが唯一の焦点面内の細胞を傷つけることなく、上記のセルとレーザービーム内にある下にはありません。レーザーが最適に設定し、z軸内の最大の焦点に合わせて調整されていることを実験前に確認してください。どのセルを知るために、実験を行う前に、レーザー損傷の深さを決定するのが最善ですsは正確に怪我をされます。 <ol class="lalpha" start="3"><li>蛍光標識された細胞を標的とした場合、通常の使用のために、明や蛍光：実験を視覚化するために必要な光を設定します。 </li><li>けがの所望の程度に応じて対象地域、またはいくつかのパルスにレーザ光のパルスを送信します。 </li></ol> 4。傷つけおよびリカバリの分析<ol class="lalpha"><li>必要に応じて、レーザー損傷の手順は、ライブ（ 例えば Metamorphソフトウェアのストリーム取得オプションを使用して）6を記録することができます</li><li>すぐに負傷した後、胚はピンセットでアガロースから優しく除去し、回復のために卵水に戻さなければなりません。 </li></ol> 注2：筋活動は、（水泳やけいれん）骨格筋の回復のために特に重要です。胚がtricaine曝露と硬いアガロース埋め込みのために移動することはできませんので、recommeないあまりにも長い間、スライドとカバーガラスの間に胚を維持するために又、作業。 <ol class="lalpha" start="3"><li>示されるように、回復した後、胚は、光、蛍光または共焦点顕微鏡によって固定されており、必要に応じて分析することができます。 </li></ol>

<ol>
	<li>Charge, S. B., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+regulation+of+muscle+regeneration.">Cellular and molecular regulation of muscle regeneration.</a> Physiol. Rev. 84, 209-238 (2004).</li><li>Steffen, L. S. <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+runzel+muscular+dystrophy+is+linked+to+the+titin+gene.">The zebrafish runzel muscular dystrophy is linked to the titin gene.</a> Dev. Biol. 309, 180-192 (2007).</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>Kawahara, G., Serafini, P. R., Myers, J. A., Alexander, M. S., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+dysferlin+by+morpholino+knockdown.">Characterization of zebrafish dysferlin by morpholino knockdown.</a> Biochem. Biophys. Res. Commun. 413, 358-363 (2011).</li><li>Behra, M., Etard, C., Cousin, X., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+mutants+to+identify+secondary+target+effects+of+acetylcholine+esterase+inhibitors.">The use of zebrafish mutants to identify secondary target effects of acetylcholine esterase inhibitors.</a> Toxicol. Sci. 77, 325-333 (2004).</li><li>Otten, C., 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=Xirp+proteins+mark+injured+skeletal+muscle+in+zebrafish.">Xirp proteins mark injured skeletal muscle in zebrafish.</a> PLoS One. 7, e31041 (2012).</li><li>Roostalu, U., Str&#228;hle, U. <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+imaging+of+molecular+interactions+at+damaged+sarcolemma.">In vivo imaging of molecular interactions at damaged sarcolemma.</a> Dev. Cell. 22, 515-529 (2012).</li><li>Stickney, H. L., Barresi, M. J., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somite+development+in+zebrafish.">Somite development in zebrafish.</a> Dev. Dyn. 219, 287-303 (2000).</li><li>Stellabotte, F., Dobbs-McAuliffe, B., Fernandez, D. A., Feng, X., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+somite+cell+rearrangements+lead+to+distinct+waves+of+myotome+growth.">Dynamic somite cell rearrangements lead to distinct waves of myotome growth.</a> Development. 134, 1253-1257 (2007).</li><li>Choi, W. Y., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration.">Cardiac regeneration.</a> Curr. Top. Dev. Biol. 100, 319-344 (2012).</li><li>Stewart, S., Stankunas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+dedifferentiation+provides+replacement+tissue+during+zebrafish+fin+regeneration.">Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration.</a> Dev. Biol. 365, 339-349 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. , 203-253 (1995).</li></ol>

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レーザー媒介傷害はゼブラフィッシュ胚で制御された条件の下で再生を勉強するために、細胞を切除することにより、所望の大きさの傷を負わせるための強力な方法です。特に、細胞を正確に標的とすることができる（ 図2）と傷害エリアとタイミングの両方を制御することができる。その後、損傷部位と再生のプロセスが容易に、監視記録された（ 図3）と（ <strong...

<table border="1"><tbody><tr><td>加熱ブロック</td><td></td><td></td><td></td></tr><tr><td>ペア＃5鉗子</td><td>デュモン</td><td></td><td></td></tr><tr><td>スライドガラス</td><td>メンツェル</td><td></td><td> 76×26ミリメートル</td></tr><tr><td>カバースリップ</td><td>ロート</td><td></td><td> 50×24ミリメートル1位</td></tr><tr><td>ワセリン</td><td></td><td></td><td></td></tr><tr><td>実体顕微鏡</td><td>ライカ</td><td></td><td> MZFLIII </td></tr><tr><td> Micropointレーザー</td><td>アンドール·テクノロジー</td><td></td><td></td></tr><tr><td>蛍光顕微鏡</td><td>ツァイス</td><td></td><td> Axioplan II </td></tr><tr><td> Metamorphソフトウェア</td><td>分子デバイス</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 試薬 <ul><li>低融点アガロース（＃50081、ロンザ） </li><li> Tricaine原液：400mgのTricaine（＃-5040、シグマアルドリッチ）/ 100ミリリットルのdH 2 O pH9.0の</li><li> E3の媒体（5 mMのNaCl、0.17のKCl、0.33 mMのCaCl 2、0.33 mMのMgSO 4）し、 </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

様々な実験的アプローチがmyotoxin注射（ブピバカイン、心臓毒性またはnotexin）、筋移植（除神経·脈管切除再生を誘導した）、激しい運動ではなく、ネズミ筋ジストロフィーのモデルを含む、筋肉の再生を研究することを目的として筋損傷を誘発するためにマウスに使用されてきたmdxマウス（これらの方法の見直しのための1を参照）など。ゼブラフィッシュでは、遺伝学的ア ​​プローチは、筋ジストロフィーの表現型（例えばrunzel 2またはsapje 3など）を展示し、筋ジストロフィーに関連する遺伝子の発現をブロック4オリゴヌクレオチド、アンチセンスモルフォ変異株が挙げられる。加えて、化学的ア ​​プローチは、それによって最終的に筋ジストロフィー5につながるhypercontraction、その結果、また、ガランタミン、化学阻害する化合物とアセチルコリンエステラーゼなどが可能です。しかし、遺伝的および薬理学的アプローチグラム物理的に与えた傷害の程度がより簡単に空間的にも時間的に制御されているのに対し、1 enerally、個々の内のすべての筋肉に影響を与えます。ローカライズされた物理的な損傷は、内部統制として、反対の筋肉の評価を可能にする。別のグループは最近、非常に局所的に個々の胚ゼブラフィッシュの筋肉の細胞膜にダメージを与える二光子レーザー（822 nm）での使用を報告しながら、実際、我々は最近、ゼブラフィッシュ胚6に骨格筋の再生を研究するためにレーザーを介する細胞アブレーションを使用細胞7。 ここでは、ゼブラフィッシュ胚における骨格筋細胞傷害に対してmicropointレーザー（アンドールテクノロジー）を使用する方法について検討する。 micropointレーザーは435 nmの波長での標的細胞の切除に適している高エネルギーのレーザーです。レーザー顕微鏡を同時にfで使用できるような方法で顕微鏡（我々のセットアップで、ツァイスの光学顕微鏡）に接続されているまたは試料上および創傷（明視野、蛍光）の効果を可視化するためのレーザー光を当てた。レーザーパルスを制御するためのパラメータは、波長、強度、パルス数を含む。 その透明性と外部の胚発生に起因して、ゼブラフィッシュ胚では、レーザ誘起損傷の両方のためとその後の回復を研究するために非常に適しています。 1〜2日後の受精、体節、骨格筋細胞が徐々にテール8,9にトランクから体節形成の進行による後方から前方に成熟する。これらの段階で、胚は、自発的にけいれんや水泳を開始します。ゼブラフィッシュは、最近、組織再生の研究のための重要な脊椎動物のモデル生物として認識されている、組織のように多くの種類（心臓、神経、血管など）が成人ゼブラフィッシュ10、11の損傷後に再生することができる。

レーザー媒介損傷が固定化された1日齢の胚で行われた。 図1に示すように、いくつかのレーザーパルスは、通常、体節の境界との間に張り渡されたときに、損傷を受け、コイル、アクチンに富んだ筋原線維で容易に認識でき、小傷を生成することができます。レーザーパルスの数値が高いしかし、ほとんどの筋原繊維が破壊された大規模な損傷を受けた体節ブロックになります?...

Watch this Scientific Journal Video about Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle at JoVE.com

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

ここで紹介する方法は、高エネルギーのレーザーパルスと時間でこれらの損傷とその回復のその後の分析でライブゼブラフィッシュ胚の正確な傷害を含む。我々はまた、遺伝的に単一標識又は骨格筋細胞のグループが中に、レーザー光誘起損傷後に追跡することができる方法を示しています。

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 ...

laser-inflicted-injury-of-zebrafish-embryonic-skeletal-muscle

Research

JoVE Journal

Biology

10.4K ビュー.  Max Delbrück Center for Molecular Medicine. ここで紹介する方法は、高エネルギーのレーザーパルスと時間でこれらの損傷とその回復のその後の分析でライブゼブラフィッシュ胚の正確な傷害を含む。我々はまた、遺伝的に単一標識又は骨格筋細胞のグループが中に、レーザー光誘起損傷後に追跡することができる方法を示しています。

ビデオ: Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

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 ...

生物学

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 ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨格筋からのミトコンドリア単離

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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.

複屈折およびタッチ呼び起こすエスケープ応答アッセイによる幼虫のゼブラフィッシュ骨格筋の欠陥の分析

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...