This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

全ての動物手順は、三重大学の倫理委員会によって承認された、と「動物の愛護及び管理に関する法律「日本動物福祉の規制（日本の環境省）に従って行われ、国際的なガイドラインを遵守しました。 ニードルの作製注：全ての実験は、麻酔下で行い、すべての努力が苦痛を最小限にするために行われました。安楽死のために、魚を≥20分間（≤4℃で5部の氷/水1部）を氷水浴中に浸漬しました。 <ol><li>針プラー（ 図1A）と1.0 mmの外径のガラスキャピラリーを引いて、ガラスマイクロキャピラリー針を準備します。 </li><li>斜めに細かいハサミ（ 図1B）を使用して、針の先端をカットします。 200ミクロン（ 図1C） -理想的な先端の直径は約100である必要があります。先端diamete場合rが狭すぎる、血液が針に入ることはありません。 </li><li> 5mg / mlの濃度に生理食塩水でヘパリンを溶解します。 </li><li>吸引管アセンブリのノーズピース端でのプレカット針を置き、口の中にマウスピースを保持する、または電球ディスペンサーに針を接続します。ヘパリン溶液に針の先端を浸し、吸引により針をヘパリンで治療すると、溶液（ 図1Dおよび1E）を吹き抜けます。 注：吸引管アセンブリは、ゼブラフィッシュ精子の凍結保存の16のために使用されてきました。ロングゴムチューブは、口の中に飛んで任意の血液を停止することができます。チューブの途中にフィルタを設定すると、危険を避けることができます。 </li><li>少なくとも1時間、10-cmのシャーレと空気乾燥でヘパリン処理針を保管してください。針の多くは、事前に製造することができます。 </li></ol> 2.麻酔<ol><li> 2（2-フェノキシエタノール、100μlの魚の水200mlを混合することにより、小さなプラスチックケースで麻酔液を準備します-PE）。麻酔薬の最終濃度は500ppmです。 注意！ 2-PEは、迅速な効果があります。 </li><li>循環系からの魚（AB株）の所望の数を削除します。 </li><li> 2分（ 図1F） - 1のための麻酔薬に魚を転送するためにネットを使用してください。 1分以内に急速な蓋の動きを徐々に、泳ぐ、水平にあえぎを胸ひれを広げ、持っている魚を観察します。 </li><li>時が経つにつれ、魚がケースの底に横たわっ観察し、最終的に水泳を停止します。魚が息をのむために停止し、蓋の動きが遅いとき麻酔の外科的平面に到達します。この時点で、魚は採血のための準備ができています。 </li><li>スキマーを使用すると、2-PEから麻酔した魚を持ち上げ、ゆっくりと麻酔薬（ 図1G）に浸したペーパータオルの上に置きます。目の乾燥を防ぎ、やさしくするために別の乾いた柔らかいティッシュペーパーを使用することも2-PE液に浸した柔らかいティッシュペーパーで魚の頭をカバー体表面を乾燥。 </li></ol> 3.採血<ol><li>吸引チューブアセンブリ（または電球ディスペンサー）のノーズピース端でのヘパリン処理針を置き、口の中に吸引管アセンブリのマウスピース端部を保持します。 </li><li>ノーズピース端と一緒に針をつかみ、慎重に針の先端に干渉スケールを除去。採血部位へ45°の角度 - 30に針を挿入します。消化管（ 図1H）の穿刺を避けます。バルブディスペンサーを用いた場合には、親指と中指とバルブを押して、上記のように針を挿入し、最初の指で電球の先端の穴をブロックします。 注：採血用サイトでは、背側大動脈の領域で肛門に体軸と後部に沿っています。背側大動脈（DA）と後部主静脈（PCV）は、脊椎に腹だけ（ 図2）です。 </li><li>吸うために開始針が脊椎に接触したときに感じられる吸引管アセンブリの端部がマウスピース。血液が上昇しない場合、血液の流れを促進するために手で微妙に針の先端を移動させます。血液が針に上昇されると、すぐに揺れ停止し（ 図1I）優しく吸うことに注意してください。バルブディスペンサーを用いた場合には、血液を吸引するためのバルブの圧力を解放します。 </li><li>血液がゆっくりため動脈圧の可能性が高い、吸引することなく、拍動性の方法で針に上昇します確認します。したがって、針が正しく動脈を貫通した場合吸引する必要はありません。 </li><li>血液の適切な量を収集した後、吸引を停止します。魚から針を外し、任意の出血を止めるために、柔らかいティッシュペーパーを使用して穿刺部位を押してください。指の圧力の20秒（ 図1J） -約10後の出血の停止を確認します。 </li><li>出血が停止した後、直ちにCLEに戻し魚を転送します暖かい水（〜28℃）のタンク。それは泳ぐことを始めるまで優しく鰓に向かって水を旋回することによって回復する魚を助けます。 注意：2リットルタンクで5 postsampling魚まで保ち、魚に酸素する循環システムにタンクを接続します。魚の水への抗生物質の添加は不要です。通常の住宅と給紙に魚を維持します。 </li><li>パラフィルムの一部（ 図1K）のクリーン領域に針から血液を追放。 </li><li>すべての市販のハンドヘルドグルコメーター（ 図1L）を使用して、血糖値を測定します。 注：血糖計は、グルコースデヒドロゲナーゼ、フラビンアデニンジヌクレオチドの電極を使用し、0.6μlのサンプル量が必要です。 <ol><li>メーター内に完全テストストリップを挿入し、直接血液滴をタッチします。自動的にテストストリップに採血を確認し、表示領域の血糖結果5秒を取得します。結果を記録し、テストストリップを廃棄します。使用各測定のために新しいテストストリップ。 </li></ol></li><li> （オプションのステップ）は、ピペッティングにより血液の正確な量を取り、さらなる分析（ヘモグロビン、トリアシルグリセロール（TG）、総コレステロールなど ）のためのマイクロ遠心チューブに血液を移します。必要に応じて、生理食塩水で1魚から全血を希釈します。室温で680×gで3分間、血液サンプルを遠心分離し、血漿を採取します。新しいチューブにプラズマを転送します。この時点で、それは、生化学的分析に使用する準備ができています。 </li></ol>

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The authors have nothing to disclose.

ここでは、直列大人のゼブラフィッシュから血液を取得するための詳細なプロトコールを提示します。この方法は、実施するのは簡単であると私たちは日常的に研究室で使用します。この採血方法は、ゼブラフィッシュの背側大動脈にガラスキャピラリ針の挿入に基づいています。この手順の間、それは背側大動脈を検索するための基準であるため、脊椎を切除しないように注意することが?...

その臓器や遺伝学は人間1,2と同様であるため、ゼブラフィッシュは、ヒト疾患の貴重なモデルとして増加し人気を集めています。発生生物学の分野では、多くの研究は、ゼブラフィッシュとヒトのショーは造血3、止血4,5、および骨髄造血6の類似性をマークしていることを実証しました。大人のゼブラフィッシュはまた、このモデル生物を共有ので、ヒトの疾患において破壊と共通の経路を免疫学7、神経変性8および肥満関連疾患9を研究するために使用されています。肥満及び肥満関連疾患（糖尿病、脂肪肝と非アルコール性脂肪肝炎およびアテローム性動脈硬化症）のために、ゼブラフィッシュ血糖および脂質のレベルは完全にいくつかのトランスジェニックおよび食餌性肥満モデル10-13に研究されてきました。 個々の動物からの反復採血は動物の使用量を削減し、12月になります個人間の違いをrease。しかし、繰り返しサンプルの収集は、その比較的少量の血液量と簡単にアクセス血管の欠如のようなゼブラフィッシュのような小動物では技術的に困難です。これらの方法は、自分の欠点を持っているが、ゼブラフィッシュからの1回の血液採取のためのいくつかの方法は、組織損傷および制限された血液量に関連する、致死性を含む、開発されています。例えば、1〜5μlの血液は背側大動脈5の領域の長さは0.3程度センチ横切開から収穫することができます。胸ガードルを切断することにより、はさみで断頭は、5〜10μlの血液10を収集することができます 。別の便利な採血方法は尾切除14です。心臓穿刺は、同じ魚から繰り返し採血のための1つの潜在的な代替方法ですが、この手順で取得した、非常に少量（約50 NL）はperforすることができます分析の数を制限11のmed。したがって、新しいプロトコルは、この生物はヒト疾患のための標準的なモデル生物であることをするために必要な重要な進歩となるであろう繰り返さ非致死性の血液サンプリングを、有効にするために必要とされています。この技術は、薬理学的応答、分子診断のためのバイオマーカー、予後の決意の発見、ならびに代謝性疾患、変性疾患および悪性疾患のいくつかの種類のような様々な疾患の監視を試験することを可能にします。 したがって、我々は、ゼブラフィッシュシリアル15から血液を得るための低侵襲的方法を開発しました。ここでは、視覚的に手順を示し、この技術の詳細なプロトコルを提供します。空腹時血糖、ヘモグロビンを含む様々なパラメータに基づいて、正常値をこの方法を用いて、そして健康な成人のゼブラフィッシュの血液中の脂質を評価しました。さらに、我々はまた、この方法は、mで一連の試料を必要とする研究に適しているかどうかを評価しましたオーバーフィード実験中血糖値の時間変化をonitoring。

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		<tr>
			<td>Glass capillaries with filament</td>
			<td>Narishige</td>
			<td>GD-1</td>
			<td>1.0-mm-outer-diameter.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>To produce the needls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>081-00136</td>
			<td>For heparinization</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Drummond</td>
			<td>2-040-000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Bulb dispenser</td>
			<td>Drummond</td>
			<td>1-000-9000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-12075</td>
			<td>For anesthetizing the fish</td>
		</tr>
		<tr>
			<td>DRI-CHEM3500V</td>
			<td>Fujifilm</td>
			<td>-</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>DRI-CHEM Slides</td>
			<td>Fujifilm</td>
			<td>Hb-WII</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>Glutest Neo Super</td>
			<td>Sanwa Kagaku Kenkyusho</td>
			<td>-</td>
			<td>For bood glucose measurement</td>
		</tr>
		<tr>
			<td>Wako L-type TG kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>464-44201</td>
			<td>For TG measurement</td>
		</tr>
		<tr>
			<td>Wako L-type CHO kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>460-44301</td>
			<td>For total cholesterol measurement</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan Packaging</td>
			<td>PM996</td>
			<td>To expel the blood on</td>
		</tr>
	</tbody>
</table>

repeated blood collection for blood tests in adult zebrafish

反復採血は、動物実験で実行される最も一般的な技術の一つです。しかしながら、ゼブラフィッシュからの血液採取のための非致死プロトコルが確立されていません。ゼブラフィッシュからの採血のための従来の方法は、横切開、断頭とテールアブレーションとして、致死的です。したがって、私たちはここで小説「繰り返し」採血方法、および現在のこの手順を概説し、詳細なプロトコルを開発しました。この方法は、低侵襲性であるため、同じ個体から繰り返し採血を可能にする、ゼブラフィッシュのための非常に低い死亡率（2.3％）が得られます。採血の最大量は、魚の体重に依存します。間隔で繰り返し採血用のボリュームが血中ヘモグロビンの測定により評価した2週間毎に、体重の≤0.4％毎週または≤1％であるべきです。さらに、ヘモグロビン、空腹時血糖、血漿トリアシルグリセロール（TG）、総C男性と女性の大人のゼブラフィッシュにおけるholesterolレベルを測定しました。また、食餌性肥満におけるグルコース代謝の調節不全を調査するためにこの方法を適用しました。この血液採取法は、グルコースおよび脂質代謝およびヒト疾患モデル生物としてゼブラフィッシュの使用を増加させる血液学の研究を含む多くのアプリケーションを可能にします。

この血液採取法は、ゼブラフィッシュ（&lt;1 mmの穿刺;図1J）に最小の損傷を引き起こし、2.3％の非常に低い死亡率をもたらします。我々は、単一の魚から収集し、その体重（ 図3）への関係を評価することができた血の最大音量を検討しました。我々は、収集した最大血液量が直線的に体重（R = 0.813）と相関することを見出しました。個々の魚（体重= 1.07...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

アダルトゼブラフィッシュにおける血液検査のための反復採血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.8K ビュー.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

ビデオ: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

アメリカカブトガニ、カブトガニポリフェムスから採血

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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

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

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

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

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

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

大人のゼブラフィッシュの生化学的解析のための採血

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

血漿タンパク保存用採血キットからゲノムDNAを抽出するために実用的で新手法

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

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 ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

キナーゼ阻害剤に対して耐性変異のスクリーニングおよび検証のための方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

フローサイトメトリーを用いて細胞外小胞の分析のための技術

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...