これらのメソッドは、JT（DE016678）にデポール大学研究評議会及びNIH / NIDCR R01助成金によって提供されていたJTの支援の研究室における研究の休暇中にEELによって開発されました。我々は感謝キャロラインハンター、CMRCゼブラフィッシュの中核施設とリーナPawluczuk、私たちの学部研究室の助手の動物のケアの技術者の努力を認める。

ゼブラフィッシュの飼育ゼブラフィッシュは、標準的な方法1に従って収容し、飼育されています。後期幼虫期間を過ぎて、ゼブラフィッシュの発生段階は、尾鰭（posteriormost hypuralプレート）のベースに上唇のanteriormost点から、その標準的な長さ（SL）、またはミリメートルの直線距離で測定することができる2。約一ヶ月後の受精（10〜12ミリメートルSL）で、鼻腔と上顎barbelsはそれぞれ、嗅覚ピットに近いと上顎の後方の角の透明上皮芽として表示されます。ゼブラフィッシュが成熟するにつれ、barbelsは狭い、ウィスカー状付属物に拡張する。上顎バーベルが大きい（2-3 mm）と、操作が簡単なので、我々のプロトコルは、この特定の付属物に適用され、同様の手法は、しかし、小さな鼻のバーベルに適合させることができる。 バーベルクリッピング<ol><li>システムの水のpHを7.0にバッファリング（3 -アミノ安息香酸エチルメタンスルホン酸）0.015％MS - 222に浸漬することにより、目的の段階（ 例えば 。、1.5〜2.5センチメートルSL、〜3-6ヶ月後の受精）で成人のゼブラフィッシュをAnaesthetize。水泳運動が停止するまで観察し、鰓の換気は、ゆっくりと規則的になります。魚のサイズに応じて、完全な麻酔が約2〜5分かかります。 </li><li>水族館魚網を使用して、シャーレ内ウェットペーパータオルの折り返し部分に時間で1〜3の魚を移す。鈍いスパチュラを用いて、向きの魚たちがお互いと左側側面上に平行になるように。手順の実行中に皮膚と鰓の潤いを保つために、最大蓋にボディ全体をカバーする魚、（鰓がカバーする）の尾部上にウェットペーパータオルの倍の一部。 </li><li>頭部を照らすために、低角度入射光を使用して、実体顕微鏡下でペトリ皿を見る。上顎の後部腹側の角（図1）から突出左上顎バーベル、の基盤に焦点を当てる。 <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="図1" /> 図1。 </li><li>しっかりと上顎の端に少し遠バーベルのシャフトを把握する。バーベルのシャフトを高めると鉗子の近位先の細いバネはさみの顎を挿入する。はさみは、安定性のための鉗子に触れることができます。最先端の上顎の端に近づくまで、バーベルのシャフトに沿ってハサミの顎をスライドさせます。バーベルのシャフトを介してカットするはさみを閉じます。切断されたバーベルは、依然として、鉗子で把持固定またはさらなる観察のために取り外すことができます。 </li><li>すぐに表在性真菌感染を制御するために、メチレンブルーの一滴を含むクリーンなシステムの水（〜500ml）の小さな水槽に魚を移す。魚が一晩回復することができます。翌朝、飼育システムに魚を戻す。バーベル再生には、2週間後に最大となる。 </li></ol>マッチドバーベルペアの寒天埋め込み<ol><li>適切な安楽死法による魚を収集し、4℃で一晩攪拌しながら緩衝生理食塩水（PBS）、4％の50 mlチューブパラホルムアルデヒド - リン酸に修正。固定量は、組織の少なくとも10倍のボリュームでなければなりません。固定後、承認された容器にパラホルムアルデヒド廃棄物を処分し、PBSの、いくつかの変更で徹底的に組織をすすいでください。 </li><li> 250mLのガラスフラスコに蒸留水で2％アガロース（T M〜37℃）150mLのを準備します。完全に溶けるまで電子レンジやホットプレートで熱は、定期的にかき回す。 50〜60℃の水浴中で融液を平衡化させます。 </li><li>しっかりとバッファーチャンバーの側面に対して、小さなガスケットゲルの金型を（〜100mLのゲル体積10 × 10cm）に配置することにより、標準的なゲル電気泳動のリグを組み立てます。 2月4日、小歯サンプル櫛（4 × 1.5 mm）を追加します。 </li><li>レベルの表面に、非常に浅い井戸を形成し、ちょうど櫛のベースをカバーするために鋳型に溶けたアガロースを注ぐ。すぐに温水浴中に残ってアガロースを戻す、これは組織を（9-12ステップ）を埋め込むために後で使用されます。アガロースと同じ温度にピペットを保つためにフラスコに長いガラスパスツールピペットを置く。 </li><li> 20〜30分硬化にゲルを許可する。櫛を削除し、バッファーチャンバーから硬化ジェルを含むゲルの金型を削除します。ゲルの外にスライドしないようにしっかりと研究室のテープとゲル型のオープンサイドをテープ。追加のアガロースは（ステップ13）後で追加できるように、このテープは、アガロースの表面上に数mmを拡張する必要があります。 </li><li>実体顕微鏡のステージ上でゲルの金型を置きます。空井戸を拡大し、フォーカスに端をもたらす。 </li><li>ウェルに隣接アガロースの表面に、水または固定バーベルの標本（ 例えば、再生成および制御）を含む緩衝液の滴をピペット。 USIngは、虫ピンを曲げて井戸に湿った標本をドラッグし、一番下にプッシュ。オリエントは、バーベルシャフトの互いに平行と同様の反対側の長辺に対して位置合わせ。表面張力は、位置で組織を保持するのに役立ちます。 </li><li>ときに埋め込むことができ、微細なピ​​ペットチップまたは井戸から余分な水分を除去するために実験室の組織の角を使用。組織が乾燥させないようように迅速に働く。 </li><li>温められたガラスパスツールピペットを用いて、軽く場所にバーベルの組織をシールするために標本とその周辺で新鮮な溶融アガロースをピペット。入れすぎないでください。アガロースが固まる前に、虫ピンで土壇場の調整を行う。 </li><li>よく隣の番号の紙標本のラベルを配置し、アガロースの小さなドロップで固定します。 </li><li>繰り返します標本の各ウェルのために6〜10を繰り返します。 </li><li>画像のキャリブレーションのために必要に応じて、印刷されたマイクロメートルのスケール（ 例えば、と一枚の紙を挿入<a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a>空井戸の底に）。井戸の底に紙を平らにし、暖かいアガロースをかぶせます。 </li><li>標本のすべてが埋め込まれている後に、ゲルの表面は、アガロースの硬化滴で、不規則になります。平らな面にゲルを置き、上面が均一になるまで、ゆっくりと追加の溶融アガロースに注ぐ。滑らかな表面を得るために必要なアガロースの最小量を使用して、あまりにも多くのアガロースは、透過光撮影を不明瞭になります。硬化するために、この最上層を許可する。 </li><li>ゲルは、今や各バーベルのペアの総形態を記録するために実体顕微鏡のステージ上で撮影することができます。画像のキャリブレーションは、埋め込まれたマイクロメートルのスケールを撮影することによって得られる。 </li><li>ゲル全体がぬれたペーパータオルに包んで数週間、4℃でビニール袋に密封し保存することができます。 </li><li>詳しい解析方法については、barbelsのペアを含む寒天ブロックはメスまたはカミソリ刃でゲルから切り出しすることができ、パラフィン組織学用に処理または標準的な方法3,4でcryosectioning。また、個々のbarbelsは、微細な針でアガロースから解剖し、水ですすぎ、および他のダウンストリームアプリケーション（ 例えば、ホールマウント免疫組織化学または共焦点顕微鏡）用に処理することができます。 </li></ol>

<ol>
	<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=Chapter+1+General+Methods+for+Zebrafish+Care.">Chapter 1 General Methods for Zebrafish Care.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Howe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+length:+Not+quite+so+standard.">Standard length: Not quite so standard.</a> Fisheries Research (Amsterdam). 56, 1-7 (2002).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+Tissue+Samples+for+Sectioning.">Paraffin Embedding Tissue Samples for Sectioning.</a> Cold Spring Harbor Protocols. 6, (2008).</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=Ch+8.3+Agar+Embedding+for+Cryostat+Sectioning+of+Embryos+or+Larvae.">Ch 8.3 Agar Embedding for Cryostat Sectioning of Embryos or Larvae.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Hansen, A., Reutter, K., Zeiske, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+development+in+the+zebrafish,+Danio+rerio.">Taste bud development in the zebrafish, Danio rerio.</a> Dev Dyn. 223, 483-496 (2002).</li></ol>

ゼブラフィッシュ上顎バーベルは、ゼブラフィッシュのいくつかの種類の細胞の成長、維持、および再生を研究するために活用されていない組織のシステムです。バーベルの付属物がない人間のアナログを持っていないが、それに含まれる細胞の種類は非常にそれが可能な光学的に透明と解剖学的にシンプルな円筒状の構造体の皮膚、腺、メラノサイト、循環血管や神経を研究すること、保存されている。よく研究さ尾鰭と同様に、バーベルの組織は切断によって再生するために誘導することができる。解剖学的ランドマークとして、上顎の境界線を使用して、切断面は、バーベル再増殖の測定を容易に、正確に配置することができます。経験豊富な演算子の場合は、それぞれの手術は数秒しかかかりません。回復は急速であり、我々はこれまで、魚の動作には短期または長期的な影響を検出されている。手術後6ヶ月に1上顎バーベルを泳いでゼブラフィッシュ、非外科的コントロールとして食べると効果的に繁殖し、組織収集の時間に匹敵する生存率を持って、アップ。この手術の生理的影響は、1）バーベルに運ば口外味蕾がまた唇、頬およびヘッド5を含む魚の上皮の他の多くの部分、上で検出されているため、最小限であると予測、2）差別味蕾が上に表示されます72時間以内に再生成バーベルは（ルクレールら 、未発表データ。）これは、上顎バーベル大人の脊椎動物のコンテキスト内で創傷治癒、血行再建術、および神経再生を研究するための低侵襲システムです。 再生の外科的誘導した後、barbelsは形態学的測定および/または固定した組織の顕微鏡分析のための間隔で収集することができます。便利に、上顎バーベルはcryosectioning、ホールマウントの免疫組織化学、およびin situで 、パラフィン組織学を含む多くの標準プロトコルのアプリケーションを容易に、約長さ（2-3 mm）とゼブラフィッシュ胚の直径（100〜200 mm）ですハイブリダイゼーション。一緒に、これらの機能は、上顎バーベル組織修復および再生を研究するためのin vivoモデルにおいて高度に実現可能にします。

顕微鏡装置透過と入射する光ファイバ照明とステレオ解剖顕微鏡は、バーベルのクリッピングと寒天の埋め込みが必要です。 バーベルクリッピング<ul><li>魚系の水</li><li> 2ゼブラフィッシュ交差タンク</li><li> MS - 222（3 - アミノ安息香酸エチルメタンスルホン酸、シグマケミカル＃E10521 - 10G） </li><li>滅菌120 mmのペトリ皿</li><li>ウェットペーパータオル</li><li>鈍い金属へら</li><li>デュモン＃55バイオイノックス鉗子（ファイン科学のツール＃11255〜20） </li><li>ミニVannas春ハサミ（ファイン科学のツール＃15000〜00） </li><li>メチレンブルー抗真菌剤（Drs.フォスター＆スミス＃CD - 905781） </li><li>小さな水槽が魚網</li></ul>組織の収集と寒天の埋め込み<ul><li> 1Xの4％パラホルムアルデヒドリン酸緩衝生理食塩水（PF - PBS）、50 mLコニカルチューブに分注し、-20℃で凍結Cを格納するまで使用</li><li> 1Xリン酸緩衝食塩水（pHは7.27から6） </li><li> 250mLのガラス製のフラスコ</li><li>蒸留水で2％アガロース</li><li>標準的な小型のアガロースゲル電気泳動のリグ（ゲルサイズ=〜10cm角） </li><li>小歯電気泳動のサンプルコーム（櫛サイズ4 × 1.5 mm）の</li><li>温水浴（50〜60℃） </li><li>実験室でのテープ</li><li>黒は、昆虫ピン、サイズ000（ファイン科学のツール＃26000〜25）エナメル</li><li> 1-2番ピンホルダー（ファイン科学ツール＃26016〜12） </li><li> 9インチのガラスパスツールピペット</li><li>ピペットの電球またはポンプ</li><li>実験室の組織</li><li>小さな紙のラベル</li><li> （省略可能）印刷されたキャリブレーションの規模</li><li>ウェットペーパータオル</li><li> ZIP -閉鎖プラスチック袋</li><li>メス/かみそり刃（寒天ブロックをカットする） </li></ul>

methods for the study of the zebrafish maxillary barbel

Barbelsは魚類、爬虫類と両生類に見られる皮膚感覚の付属です。ゼブラフィッシュ、 ゼブラフィッシュは 、barbels短鼻ペアと長い上顎ペアの2つのペアを開発しています。バーベルの組織は、皮膚細胞、腺、味覚、メラノサイト、循環血管や知覚神経を含む外胚葉、中胚葉および神経堤由来の細胞が含まれています。ほとんどの成人の組織とは異なり、上顎バーベルは、私たちはライフサイクル全体にわたってこれらの組織型の開発と保守を視覚化できるように、光学的に透明です。 このビデオでは、上顎バーベル（約1ヶ月後の受精を開始）の早期開発を示し、成人心耳（&gt; 3ヶ月後の受精）の再生を誘導するために外科的プロトコルを示しています。簡単に言えば、麻酔魚の左上顎のバーベルは、上顎の尾側端のすぐ遠位滅菌ピンセットで上昇している。罰金、滅菌春のはさみ切断面のための解剖学的ランドマークを確立、このレベルでバーベルのシャフトをカットする鉗子に対して位置決めされる。再生成長は、と反対バーベルと比較してこの平面を基準にして測定することができます。バーベルの組織は、損傷の2週間以内に最大の再生に達し、急速に再生成されます。 再生バーベルを分析するためのテクニックは、標準的なDNAの電気泳動ゲルのウェル中で（再生成および制御）barbelsのマッチドペアを解剖し、埋め込む含まれています。埋め込まれた検体は、便利な総形態と形態計測のための実体顕微鏡下で写真撮影されている、と前のパラフィン組織学のようなダウンストリームアプリケーションに週間保存することができる、cryosectioning、および/または全体のマウント免疫組織化学。これらのメソッドは、ゼブラフィッシュの遺伝学的文脈の中で複数の細胞型の再生能力を研究するための生体組織系の小説として上顎バーベルを確立する。

Watch this Scientific Journal Video about Methods for the Study of the Zebrafish Maxillary Barbel at JoVE.com

Methods for the Study of the Zebrafish Maxillary Barbel

ゼブラフィッシュ上顎バーベルは外胚葉、中胚葉および神経堤誘導体を含有する皮膚感覚器官です。重要なのは、大人のバーベルは、近位切断後に再生成することができます。このビデオでは、上顎バーベルの開発を紹介し、バーベルの標本の収集、埋め込みと下流のイメージングに続く再生を誘導するために外科的プロトコルを、示しています。

ゼブラフィッシュ上顎バーベルの研究のための方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

methods-for-the-study-of-the-zebrafish-maxillary-barbel

Research

JoVE Journal

Biology

12.4K ビュー.  DePaul University. ゼブラフィッシュ上顎バーベルは外胚葉、中胚葉および神経堤誘導体を含有する皮膚感覚器官です。重要なのは、大人のバーベルは、近位切断後に再生成することができます。このビデオでは、上顎バーベルの開発を紹介し、バーベルの標本の収集、埋め込みと下流のイメージングに続く再生を誘導するために外科的プロトコルを、示しています。

ビデオ: Methods for the Study of the Zebrafish Maxillary Barbel

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

コンピュータによって生成された動物モデルの刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

ゼブラフィッシュの視覚機能を研究するために視運動性応答を使用して

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

アダルトゼブラフィッシュへの腹腔内注射

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

腎上皮再生を研究するためにゼブラフィッシュの前腎のレーザーアブレーション

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

リジェネアダルトゼブラフィッシュテールフィンにMorpholinos の in vivoエレクトロポレーションで

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

白蝶貝からの内生菌の抽出方法<em&gt;ベミシア・タバチ</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...