マリーBirne、Areti Tsiola、JaymieベスとArelysウリベのおかげ。この作品は、CUNY研究財団、ハワードヒューズ医学研究所のグラントのサマープログラム学部生用（平）とNIH RO3 41702-00-01によって部分的にサポートされていました。研究はまた、クイーンズカレッジによってサポートされていましたし、実験の一部は、イメージングのための中核施設、クイーンズ大学で細胞分子生物学からの機器で行われた。

ゼブラフィッシュは、28時クイーンズ大学の動物施設での13個の標準プロトコル℃を用いて提起された個々の魚のペアまたはグループの交配は、あらかじめ形成し、その結果胚は、洗浄し、28℃のインキュベーターに格納する必要があります。魚の健康状態を毎日検討する必要があります。 5日後の受精（DPF）で、魚は10-15魚の密度でタンクに区切る必要がありますし、魚の施設の保育園を締結した。 15、30、45、60、90、180 DPF：ここで紹介するプロトコルのためにゼブラフィッシュは、成体の段階を経て幼虫で回収された。 1。ゼブラフィッシュの主題のコレクションと固定<ol><li>麻酔は0.2％Tricaineでネットと場所を使用して、生息地のタンクから魚を取り外します。 </li><li>安楽死させる15分間氷水で麻酔した魚を置きます。 </li><li> 1Xリン酸緩衝食塩水（PBS）で新鮮な4％パラホルムアルデヒド（PFA）を作る。 </li><li> 4℃で一晩室温で1-2時間、4％PFAで収集された魚をインキュベートしますパラフィルムでラップされたペトリ皿で​​C。これは魚のフラットを保持し、後で解剖が容易になります。 </li><li>固定後、ツイーン（PBT）をPBSで2回すすいでください。魚は、最大10日間4℃でPBTしてくださいすることができます。 </li></ol> 2。魚や解剖の測定<ol><li>ペトリ皿の半分に代わり、単一の魚は新鮮なPBSで満たされ、その右側に置きます。 </li><li>デジタルミリメートルキャリパー（図1）を使用していないテールフィンを含む、鼻から尾の付け根（尾柄部）への魚の測定の長さ、、。これは標準の長さ（SL）です。 </li><li>それぞれの魚は後で分析するために追跡する個々の魚と結果の解剖、心臓を可能にするために数値/文字の組み合わせを割り当てることができます。親の株式の数、固定時の年齢、SLの測定を含むデータは、スプレッドシートに入力され、これらのラベルに基づいて組織された。 </li><li>割り当てられている追跡番号を持つ郭清、ラベルPCRチューブストリップの前に、さらなる分析のために適切なPBTまたは他の溶液を満たす。 </li><li>オリエントペトリ皿の上の魚腹側眼と鰓（図2）の間に頭部を保持して鉗子で体を安定化しながら。 </li></ol> 小さ ​​な魚（12ミリメートルSLよりも小さい）。 <ol start="4"><li>ピンホルダーの急激な鉗子を使用して）、またはマイクロ針、心を明らかにするためにボディから胸筋とフィンを削除します。解剖時に鉗子の一定の動きは、魚を移動するPBTの電流を引き起こす可能性があります。これは、このように、マイクロ針が胸筋とフィンを削除し、体腔を開くために使用できる、小型魚のために特に問題です。 </li><li> a）はゆっくりと吹き抜けの下からキャビティから心臓をえぐり出すために鉗子を使用する。交互に、いくつかの魚では静かに球根状の外に引き出し、心の残りが続くことによって心臓を削除することができます。それは簡単に破損することができるようにこのテクニックを使用している場合はアトリウムの位置に注意してください。心に付属している余分な組織がある場合、それは素晴らしいですし、後にクリアすることができます。 </li><li>魚はそのようなTG（myl7として蛍光心臓マーカーが含まれている場合はA）：GFP）twu34を 、解剖用蛍光スコープを使用し、蛍光（図3）により、心臓の除去を確認してください。 </li><li>心臓後）は空洞から削除され、心臓の外から余分な非心臓組織と心外膜ライニングを削除するには、心臓やマイクロ針を保持するために鉗子を使用する。 </li></ol> 大きな魚（12ミリメートルSLよりも大きい）。 <ol start="4"><li> b）の春は、マイクロはさみを処理する使用は、深さ1mm未満の三切開して：1 - 鰓2を介して横方向のカット - 前腹3での横方向のカット - カット（図4）の両方を接続する腹側矢状カット。 </li><li> B）鋭いピンセットを使用して体腔を開くためにボディから胸筋とフィンを削除し、心膜（図5A）の銀色の組織を明らかにする。この心膜組織を切除すると心臓が（図5B）見えるようになります。 </li><li> b）の中心に優れた位置球根状の動脈に接続されている動脈を、カットするマイクロはさみを使用してください。この動脈が切断されると、アトリウムの下に鉗子先端を入れて、空洞の中心をかき出すために鉗子を使用する。心に付属している余分な組織がある場合、それは素晴らしいですし、後で削除することができます。 </li><li>心が空洞から削除されたらb）に、余分な、非心臓組織を除去するために心臓やマイクロ針を保持するために心を保持し、余分な組織を削除するには2つの鉗子を使用して、または鉗子の一組。そっとティッシュをこすることによって、必要に応じて、その黒い色素沈着によって明らか心外膜ライニングは、心臓の外側から削除することができます。 </li></ol> 3。心の写真<ol><li> 23グラム/ L寒天培地をペトリ皿を準備し、PBSで固化した寒天をカバー。 </li><li>鉗子を使用して、心臓が撮影のために所望の配向で休むことができる寒天でよくすること。 </li><li>リムーバブルしっかりと鉗子と球根状の先端部を保持することによってPCRチューブからのEは心臓。寒天料理で心を置きます。 </li><li>写真の寒天のオリエントの中心。サンプルは、全ての可能な方向（図6）に、各ピクチャの後に回転させることができる。 </li><li>心を撮影するためにオーバーヘッドが光と短い露光時間を使用してください。最高の写真を得るために、必要に応じて露光を用いて光質のための調整を行います。 </li></ol> 4。代表的な結果： よく解剖クリーン心臓の例を図6に複数の向きで表示されます。それぞれの心には、全体的な形態のいくつかの違いがあるわけだが、心はそのまま心室、心房と球根状の動脈を含む、完全なはずです。全体的な形態におけるこの変化のいくつかの例を図7に示されています。黒い斑点心外膜組織はまた、心臓から削除できます。 <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="図1"/> 図1鼻からデジタルミリメートルキャリパーを使用して、尾柄部に魚を測定します。 <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="図2"/> 図2：目と鰓の間にピンセットで魚を持ちます。 <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="図3"/> 図3のようTG（myl7：GFP）などの心臓特異的蛍光遺伝子組換えマーカー。ここに示されているtwu34は 、、小さな魚から摘出心を視覚化に役立ちます。 <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="図4"/> 図4胸筋と皮膚を除去するための3つの切開部：1 -鰓2を介して横方向のカット-前腹3での横方向のカット-両方のカットを結ぶ腹側矢状カット。。 <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="図5"/> 。球根状の動脈（BA）、心室（V）、およびアトリウム（）： 図5ブラック文字は、心臓の室にラベルを付けます。 大文字体腔は心膜の銀色の組織を表示する開いている。 B -後に心膜を除去し、心は目に見えると簡単に削除です。 <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="図6"/> 心臓の図6。異なる方向が表示されます。球根状の動脈（BA）、心室（V）、およびアトリウム（）：黒い文字は、心臓の室にラベルを付けます。 、（）前方後方（P）、背側（D）、腹側（V）、左（L）、右（R）：グレーの矢印は、体内での正常な位置への参照の中心の位置と方向を示している。スケールバーは400μmを示しています。 心臓の腹ビュー 心臓のB -背ビュー 左サイドからのC -側面図 右側からD -側面図 <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="図7"/> 図7。異なるサイズの心臓の形態が示されています。球根状の動脈（BA）、心室（V）、およびアトリウム（）：黒い文字は、心臓の室にラベルを付けます。スケールバーは400μmであることを示した。 AC -ハーツ未満12ミリメートルSLを持つフォームの魚 DF -ハーツ12ミリメートルを超えてSLを持つフォームの魚

<ol>
	<li>Ahmad, N., Long, S., Rebagliati, 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+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+zebrafish+heart:+from+left-right+axis+specification+to+epithelial+tissue+morphogenesis.">Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis.</a> Dev Biol. 330, 213-220 (2009).</li><li>Glickman, N. S., 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=Cardiac+development+in+zebrafish:+coordination+of+form+and+function.">Cardiac development in zebrafish: coordination of form and function.</a> Semin Cell Dev Biol. 13, 507-513 (2002).</li><li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</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>Beis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+cellular+analyses+of+zebrafish+atrioventricular+cushion+and+valve+development.">Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.</a> Development. 132, 4193-4204 (2005).</li><li>Lepilina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+epicardial+injury+response+supports+progenitor+cell+activity+during+zebrafish+heart+regeneration.">A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration.</a> Cell. 127, 607-619 (2006).</li><li>Wills, A. A., Holdway, J. E., Major, R. J., 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=Regulated+addition+of+new+myocardial+and+epicardial+cells+fosters+homeostatic+cardiac+growth+and+maintenance+in+adult+zebrafish.">Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish.</a> Development. 135, 183-192 (2008).</li><li>Parichy, D. M., Elizondo, M. R., Mills, M. G., Gordon, T. N., Engeszer, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Dev Dyn. 238, 2975-3015 (2009).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, 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=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</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=The+Zebrafish+Book,+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio).</a> , (1995).</li></ol>

利害の衝突は宣言されません。

後胚魚のゼブラフィッシュの心臓の解剖のためのこれらのメソッドは、無傷の心を完全に除去することができます。この手法は単純で、心臓の損傷を防止する外部削減のためのガイドを提供していますが、着実に手は小さい魚のために不可欠です。 解剖時の重要なステップは、心臓がこの嚢に直接そのまま銀色の心膜を特定することです。心膜を視覚化することはボデ?...

<table border="1"><tbody><tr><th>材</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>パラホルムアルデヒド</td><td> EMD </td><td> PX0055 </td><td> PBSで.04g/mL </td></tr><tr><td>鉗子＃55 </td><td> FST </td><td> 11295〜51 </td><td></td></tr><tr><td>マイクロはさみ</td><td> FST </td><td> 15005〜08 </td><td></td></tr><tr><td>ピンホルダー</td><td> FST </td><td> 26016〜12 </td><td></td></tr><tr><td>マイクロ針</td><td> FST </td><td> 10130〜10 </td><td></td></tr><tr><td>栄養寒天</td><td> BBL </td><td> 11472 </td><td>蒸留H 2 Oで23グラム/ L </td></tr><tr><td>ペトリ皿</td><td> BDファルコン</td><td> 351029 </td><td></td></tr><tr><td> PCR 8連チューブストリップ</td><td>米国サイエンティフィック</td><td> 1402-2500 </td><td></td></tr><tr><td>デジタルノギス</td><td>フィッシャーサイエンティフィック</td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

ゼブラフィッシュは、胚心臓の開発の研究のために有益で実用的なモデル生物となっています（最近のレビュー1-6を参照）、しかし、成人の心臓の開発を通じて、後の胚調べるが70から10に制限されて動作する。成熟と老化の心の変化形態を調べることは少年と大人の心をステージングし、単離するための利用可能な技術の不足によって制限されます。魚の寿命を介して心臓の開発を分析するために、我々は多数の段階でゼブラフィッシュの心臓を解剖し、さらなる分析の11のそれらを撮影する。心臓の形態学的特徴は、容易に定量化することができると、個々の心は、さらに標準的な方法のホストで解析することができます。ゼブラフィッシュは、可変レートで成長し、成熟は、魚の成熟のゲージのようなので、後固定、我々の写真と測定の魚の長さを年齢よりも魚のサイズとのより良い相関する。このプロトコルは、30mmのSLとVL 1mm以上大きいと、大人に100μmの心室の長さの心（VL）と幼虫3.5ミリメートルの標準長さ（SL）に至るまでゼブラフィッシュの2つの異なる、サイズ依存解剖の技術を、、説明しています。仔魚と成魚では全く異なる身体や臓器の形態を持っている。幼虫は唯一大幅に小さくはない、彼らは以下の顔料を持っており、各器官は視覚的に識別することは非常に困難です。このような理由から、我々は明確な解剖の技術を使用してください。 我々は安楽死の直後に解剖の心には、次の固定を除去心と比較して心房のような非常に緩いとバルーンで、より多くの変数形態を持っていることを発見したように我々は、事前に解剖の固定手順を使用する。前の解剖に固定された魚は、 生体の形態とチャンバーの位置（データは示さず） に保持されます。私たちは、心臓全体を視覚化できるとするときに心臓の開発の初期段階で特に有用であるtwu34（12）、：加えて、デモンストレーションの目的のために、我々は、心臓（心筋）特定のGFPトランスジェニックTgは（GFP myl7）を活用する形態は、周囲の組織から少なく明瞭である。心臓の解剖は、in situハイブリダイゼーション、免疫組織化学、RNAの抽出または後の胚ゼブラフィッシュで簡単に他の分析方法で使用して心臓の開発と成熟の根底にある細胞と分子生物学のさらなる分析を行います。このプロトコルは、心臓の開発の成熟と老化の研究のための貴重なテクニックを提供します。

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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

複数の発達段階におけるゼブラフィッシュの心臓の解剖と分離のための明確な、標準化された方法が説明されています。注釈と定量化の手法についても説明します。

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 ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

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

21.4K ビュー.  Queens College, City University of New York. 複数の発達段階におけるゼブラフィッシュの心臓の解剖と分離のための明確な、標準化された方法が説明されています。注釈と定量化の手法についても説明します。

ビデオ: Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ショウジョウバエ幼虫NMJの解剖

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

アダルトゼブラフィッシュの内耳感覚上皮のグロスとファイン解剖（ゼブラフィッシュ）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

アダルトゼブラフィッシュからの臓器の解剖

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

の解剖と染色ショウジョウバエ幼虫卵巣

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

アダルトゼブラフィッシュ腎臓の解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

ゼブラフィッシュの定期的ケアとメンテナンス（<em&gt;ゼブラフィッシュ</em&gt;）研究所：はじめに

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

ゼブラフィッシュを用いた病的ヒトゲノムの in vivoモデルで

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavagingアダルトゼブラフィッシュ

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...