Name: visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish
Uploaded: 2016-12-21T13:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

LI1及びTg（kdrl：EGFP）S843：私たちは、Tgは（GFP wt1b）を贈与するための教授クリストフEnglert教授ディディエStainierに感謝します それぞれの株、及びTg（kdrl：mCherryを）提供するための台湾ゼブラフィッシュ基盤施設ci5を 。この研究は102-、科学技術（96から2628-B-029から002-MY3、101から2313-B-029から001まで、102から2628-B-029から002-MY3の台湾省からの助成金によってサポートされていました2321-B-400から018）。

倫理文：ゼブラフィッシュのすべての実験手順は、（。101-12 IRB承認NO）東海大学の施設内動物管理使用委員会によって承認され、承認されたガイドラインに従って実施しました。 3β-HSD酵素活性染色1.株価ソリューション<ol><li>トランスデヒドロ[ジメチルスルホキシド（DMSO）中に10mg / mlの]準備します。 </li><li> β-ニコチンアミドアデニンジヌクレオチド水和物（0.1 Mリン酸緩衝液中で1.2 mg / mlで、pHは7.2）を準備します。 </li><li>ニコチンアミド調製（ビタミンB3を、50 mg / mlとでH 2 O）。 </li><li> 4-ニトロブルーテトラゾリウム（70％ジメチルホルムアミド中に50mg / ml）を準備します。 </li><li> -20℃でマイクロチューブとストアに小分けし、これらすべてのコンポーネント。 注：我々の経験では、上記の全てのソリューションのアリコートの凍結融解の繰り返しは、3β-HSD活性染色の強度に影響を与えません。 </li></ol> 2. Wホールマウント3β-HSD活性染色注：3β-HSD酵素活性が早ければ28時間後に受精（HPF）として培養される場合hsd3b1 2,7のmRNA発現の発症と一致している28.5°C、でのゼブラフィッシュの胚で検出可能です。本研究では、全マウント3β-HSD活性染色プロトコルは、前述の方法8に基づいており、最大7日後に受精9に開発ゼブラフィッシュで成功するために決定されました。 ci5 15：14であり、Tg（mCherryをkdrl）を S843：腎間のステロイド産生組織の血管の微小環境を分析するため、ホールマウント3β-HSD活性染色は、Tgが（EGFP kdrlが）などの内皮特異蛍光を発現するトランスジェニック系統、に適用されなければなりません。ゼブラフィッシュ腎臓の開発を検討腎間のステロイド産生組織を分析するために、3β-HSD活性染色はアプリすることができますLI1魚 16、糸球体と前腎細管の形成が描写されている：Tgは（GFP wt1b）にエド。 <ol><li>色素形成を阻害するために、12および24 HPFの間に段階から、卵の水中の0.03％のフェニルチオ尿素（脱イオン水中の0.06 mg / Lのリーフ塩）でそれらをインキュベートすることにより、開発ゼブラフィッシュを扱います。 </li><li>で一晩、室温（RT）又は（B）の2％PFATで1時間、0.1％のTween 20（PFAT）を補充したリン酸緩衝生理食塩水（PBS）中で（A）、4％パラホルムアルデヒド（PFA）でdechorionated胚または幼虫を修正4℃または室温で4時間、続いて4℃で2時間、のために。 注意：PFAは、吸入、皮膚接触によって有毒です。それは、皮膚、粘膜、目、および上気道に破壊的です。 </li><li> PBSで10分間胚を4倍を洗って、室温で0.1％のTween 20（PBST）を補いました。 注：これは、洗浄溶液を変えながらサンプルを乾燥させないことが重要です。 </li><li>たて駅を準備反応の前にining溶液（ 表1）。別々のチューブに、準備渦、および遠心分離器の部品AとB。 注：ストレート1単一のチューブにすべてのコンポーネントを追加すると、重度の沈殿を引き起こすことになります。 </li><li> 、パートBのソリューション全体に部品Aのソリューション全体を追加染色液を調製し、よく混ぜ、すぐに固定し、洗浄した胚を含む各マイクロチューブに1ミリリットルを配布します。 </li><li>回転子または室温で穏やかに振盪ためのロッキングプラットフォームのいずれかに光と場所からそれらを保護するためにアルミホイルでマイクロチューブを包みます。 </li><li>経験的には顕微鏡を通して発色性の信号を監視することにより、反応時間を決定します。若干の沈殿は時間をかけて開発します。しかしながら、それらは、反応過程を妨害しません。 注：胚が4時間25℃で28 HPF、染色に固定するために間腎組織でクリア信号を生成します。 </li><li> PBSTで10分間の4倍を洗浄することによって反応を停止させます。私Fはさらなる免疫組織化学（IHC）分析が必要とされない、後の修正RTで60分間、4％PFATで染色された胚を。それ以外の場合は、さらに使用するまで4℃で洗浄した胚を保存します。 </li><li>全体のマウント顕微鏡検査のために、染色後固定し、そしてPBS中50％グリセロールで胚を洗浄しクリアし、背側がスライド上に向けて、それらを向けると、明視野照明下で直立顕微鏡を用いて観察します。 注：推奨される腎間の組織形態の高解像度画像は、3β-HSD活性染色とdeyolked胚の分析をフラットマウントにするには。腎間のステロイド産生組織が右卵黄嚢の上方に位置するように、間腎組織の腹側フラットマウント分析は、腎間の形態17の直接的かつ明確に観察できます。 </li></ol> 3β-HSD活性染色胚の3 IHC分析注：steroiの血管の微小環境を分析するためにdogenic組織は、トランスジェニック内皮特異的蛍光レポーターとの3β-HSD活性染色胚をビブラトーム切片化し、その後のIHC分析12,18を横断するために供されます。 <ol><li>ポスト固定<ol><li> 3β-HSD活性染色の後の修正及びPFAは、4℃で1時間、1％トリトンX-100を補った、1％トリトンX-100（PBSTx）を含むPBS中で10分間、4倍の洗浄2％で胚を洗浄RTで。 </li></ol></li><li>埋め込み<ol><li> 4％が95℃、4〜8℃でのマイクロチューブにアリコートし、店舗でPBSでアガロースを溶解させることによって低融点アガロース準備します。 </li><li> 70℃で10分間、アガロース4％低融点のアリコートを融解し、その後47℃で一定分量を保ちます。各胚を埋め込む溶融4％鋳型として働く1.5mlマイクロチューブの取り外したキャップ内に低融点アガロース、および固体になるまで冷却します。 </li></ol></li><li>ビブラトームセクショニング<ol><li>乾燥Pに紙テープの一部をスティックビブラトームのlatform。 </li><li>カミソリの刃によって金型から硬化アガロースブロックを削除してください。プラットフォーム上の紙テープに瞬間接着剤を適用し、上向きにサンプルの前方側に、紙テープにアガロースブロックを修正。それぞれ約4ミリメートルと上下ファセットの各側に8ミリメートルで、台形プリズム形状にアガロースブロックをトリム。 </li><li>スポイトを使用して、0.5％トリトンX-100（0.5％PBSTx）を補充し、冷PBSでサンプルを含むアガロースブロックをすすぎます。 </li><li>ビブラトームの取扱説明書に従って、100μmの切片にアガロースブロックをスライス。常に乾燥を防ぐために、アガロースブロックをすすぎます。各スライスが切断されると、26 G注射針を使用して、ビブラトームからそれを削除し、冷たい0.5％PBSTx（500μL/ウェル）を含むスポットプレートに移します。 </li><li>解剖顕微鏡下で確認することにより、3β-HSD活性の陽性組織切片を収集し、πによってセクションを転送冷たい0.5％PBSTx（150μL/ウェル）を含む96ウェルプレートにその端のカットオフ（開口部で約1ミリメートル、内径）とのpetteチップ。組織試料は、通常、このステップの後、アガロースブロックから解離します。 </li></ol></li><li>透過処理と洗浄<ol><li> 6X RTで0.5％PBSTxで15分間、PBS中で10分間、そしてPBS中で10分間、4倍のスライスを洗浄し、1％ウシ血清アルブミン/ 1％DMSO / 0.1％トリトンX-100（PBDTx）を補充しました。 </li></ol></li><li>ブロッキング<ol><li> RTでPBDTx 2％ウシ胎児血清（FCS）中で1時間切片をインキュベートします。 </li></ol></li><li>一次抗体反応<ol><li> 4℃で一次抗体（ 例えば 、ウサギポリクローナル抗ヒトフィブロネクチンおよびマウス抗ヒト焦点接着キナーゼ）を含むPBDTxで1.5日間のスライスをインキュベートします。 </li></ol></li><li>洗浄<ol><li>スライスはRTでPBDTxで10分間、6倍速洗います。 </li></ol></li><li>ブロッキング<ol><li> PBで2％FCSで1時間のスライスをインキュベートRTでDTX。 </li></ol></li><li>二次抗体反応<ol><li> 4℃での二次抗体（フルオロフォア結合ヤギ抗ウサギまたは抗マウスIgG）を含むPBDTxで1.5日間切片をインキュベートします。 </li></ol></li><li>洗浄<ol><li>切片を室温でPBST中で10分間、RTおよび4倍でPBDTxで10分間、6倍洗います。 注：共焦点顕微鏡分析のために、PBS中50％グリセロールでサンプルをオフにします。 </li></ol></li></ol> 4.共焦点分析<ol><li>全体のマウント胚および10X / 0.5および20X / 0.75の対物レンズを搭載した共焦点顕微鏡により断面を観察します。 </li><li>次のように蛍光および3β-HSD染色の同時検出のために、マルチトラックモードを設定：488 nmのアルゴンレーザーとGFPの検出のための505から530 nmのバンドパスフィルタと、 543 nmのヘリウム - ネオンレーザーおよびmCherryを検出するための560から615 nmのバンドパスフィルター。そして、3β-HSD染色の検出のための光チャネルを送信しました。ピンホールのサイズは、1 Airey単位であり、解像度は1,024×1024画素です。 </li><li> 3D投影として（6μmの間隔で）のzスタックを組み立て、そして内蔵の共焦点ソフトウェアを使用することにより、突起と明視野をマージします。 </li></ol>

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The authors have no competing financial interests to declare.

3β-HSD活性の信号強度は、反応の過程にわたって増加しました。 3β-HSD活性のクリア信号が以降28 HPFからの段階のための反応の4時間後に検出されました。しかしながら、反応時間は、アッセイの目的に応じて、経験的な決意を必要とします。染色は一晩処理を必要とする場合には、わずかに青みがかった背景には、サンプル上で開発する傾向があります。この問題は、3β-HSDの活性染色の前に?...

副腎、視床下部 - 下垂体 - 副腎軸の重要な要素は、ステロイドを分泌し、ステロイドの恒常性とストレスに対する身体の応答を調整します。副腎は、カテコールアミンを合成するゾーン特異的にステロイドを分泌する外皮質、および内側髄質を含みます。硬骨魚で間腎腺はそれぞれ1-3、哺乳動物における副腎のカウンターパートであり、副腎皮質と髄質の機能的等価物であるステロイド産生腎間の及びクロム親和性細胞から構成されています。ゼブラフィッシュモデルを使用して行われた研究では、両方のステロイド産生およびクロム親和細胞系統が高哺乳類1,2のものと似た分子・細胞メカニズムによって形成されていることを報告しています。したがって、ゼブラフィッシュは、遺伝性疾患、神経内分泌制御、および視床下部 - 下垂体 - 副腎（腎間の）軸のシステム生物学を研究するための潜在的に強力なモデルです。 4,5からのアンドロステンジオンからのプロゲステロンの変換を触媒します。 3β-HSDは、ホルモン、ステロイド、すなわちプロゲステロン、グルココルチコイド、ミネ、アンドロゲン、およびエストロゲンのすべてのクラスを生合成するために不可欠です。 2つのヒト3β-HSDはHSD3B1とHSD3B2を差動6を発現しているアイソザイム。 HSD3B2の副腎皮質および生殖腺で発現されるのに対し、HSD3B1は、胎盤および末梢組織で発現されます。人間HSD3B1とHSD3B2は間腎組織および成体の生殖腺で発現されるゼブラフィッシュhsd3b1の共同オルソログです。ゼブラフィッシュhsd3b2は、その転写産物器官7の前に姿を消す母性発現する遺伝子です。ゼブラフィッシュのための全マウント3β-HSD酵素活性アッセイのプロトコルは、レヴィの方法を変更することによって開発されましたミラノらによって記載さsの。 、8硬骨魚種8の凍結切片上。そのため組織透過性と開発ゼブラフィッシュの光透過性の、ホールマウント3β-HSDの組織化学が正常に固定ゼブラフィッシュの胚および幼生のために使用され、特に区別腎間の組織を描写することができます。 この高感度かつ迅速なアッセイは、腎間の異常形態の異なる種類を実証する種々の変異体とモルファントに適用されています。腎間の3β-HSD活性は間腎組織の仕様はFf1b転写因子の特異的ノックダウンを介して破壊されると腎間の分化がFf1bの補調節因子PROX1 9,10のノックダウンによって影響されるように減少した胚には存在しません。特に、3β-HSD活性は、以下のような片目のピンヘッド 、重度の早期欠陥を有する変異体で検出することができ、どこ3β-H、 目を細めSD組織化学は、腎間の細胞遊走が11をどのように影響されるかを描きます。間腎組織の分化があっても、血液や血管系の完全な不存在下で損なわれることはありません。したがって、内皮由来の信号が整形方法を開発腎間の臓器は12,13を決定することができます。全体として、この組織化学的アッセイは、正常仕様、分化、およびゼブラフィッシュモデルにおけるステロイド産生細胞の遊走を研究するために使用されています。したがって、効率的かつ副腎および腎間の臓器障害をターゲットに任意の遺伝的または化学スクリーンのための信頼性の高いツールであるべきです。

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

34 HPFでci5胚 ：Tgは（mCherryをkdrl）; LI1：前腎腎臓糸球体とその新生血管系とステロイド合成間腎組織のcodevelopsは、3β-HSD酵素活性アッセイは、二重トランスジェニックTgは（GFP wt1b）で行った方法を決定するために、 （ 図1）。いくつかのステロイド産生細胞が正中線を横切って移行を開始し、背側のビューから突出したエッ?...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

ゼブラフィッシュにおける間腎腺は、哺乳動物の副腎の硬骨魚類の対応です。酵素活性アッセイ、開発ゼブラフィッシュでは、分化ステロイド産生細胞を検出し、このプロトコルは、3-βヒドロキシステロイドデヒドロゲナーゼ（3β-HSDΔ5-4イソメラーゼ）を実行する方法を紹介します。

腎間のステロイド合成組織とゼブラフィッシュにおけるその血管微小環境の可視化

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.9K ビュー.  Tunghai University. ゼブラフィッシュにおける間腎腺は、哺乳動物の副腎の硬骨魚類の対応です。酵素活性アッセイ、開発ゼブラフィッシュでは、分化ステロイド産生細胞を検出し、このプロトコルは、3-βヒドロキシステロイドデヒドロゲナーゼ（3β-HSDΔ5-4イソメラーゼ）を実行する方法を紹介します。 

ビデオ: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

解剖顕微鏡を使用したタイムラプスイメージングとゼブラフィッシュ腎臓の開発の分析は、光学切片のための装備します

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

発生生物学

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

ホールマウントゼブラフィッシュ胚の繊毛器官におけるリン酸化エピトープを免疫染色

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

化学

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼生ゼブラフィッシュ性腺組織の解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

ゼブラフィッシュ腫瘍異種移植片における宿主血管新生応答の生体内イメージングと定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

がん研究

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全体のマウント蛍光In Situハイブリダイゼーション、免疫組織の結合されたプロトコルを介してゼブラフィッシュにおける Multiciliated の細胞を可視化します。

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

骨組織の再生にプレドニゾロンの効果を研究する大人のゼブラフィッシュ傷害モデル

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

ブライトフィールド顕微鏡を用いた幼虫ゼブラフィッシュの肝臓サイズの定量化

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

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 ...

生物学

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

魚の目を通して糖尿病の勉強: 大人 tg(fli:EGFP) ゼブラフィッシュ網膜血管系の解析、可視化、レーザーマイクロダイ セクション

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

医学

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

ホールマウントによるゼブラフィッシュ胚標本ステンドの観察および分析のためのフラットマウントの準備<em&gt;その場で</em&gt;ハイブリダイゼーション

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

腎間のステロイド合成組織とゼブラフィッシュにおけるその血管微小環境の可視化

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解剖顕微鏡を使用したタイムラプスイメージングとゼブラフィッシュ腎臓の開発の分析は、光学切片のための装備します

ホールマウントゼブラフィッシュ胚の繊毛器官におけるリン酸化エピトープを免疫染色

幼生ゼブラフィッシュ性腺組織の解剖

ゼブラフィッシュ腫瘍異種移植片における宿主血管新生応答の生体内イメージングと定量

全体のマウント蛍光In Situハイブリダイゼーション、免疫組織の結合されたプロトコルを介してゼブラフィッシュにおける Multiciliated の細胞を可視化します。

骨組織の再生にプレドニゾロンの効果を研究する大人のゼブラフィッシュ傷害モデル

ブライトフィールド顕微鏡を用いた幼虫ゼブラフィッシュの肝臓サイズの定量化

アダルトゼブラフィッシュ腎臓の解剖

魚の目を通して糖尿病の勉強: 大人 tg(fli:EGFP) ゼブラフィッシュ網膜血管系の解析、可視化、レーザーマイクロダイセクション

ホールマウントによるゼブラフィッシュ胚標本ステンドの観察および分析のためのフラットマウントの準備

腎間のステロイド合成組織とゼブラフィッシュにおけるその血管微小環境の可視化

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解剖顕微鏡を使用したタイムラプスイメージングとゼブラフィッシュ腎臓の開発の分析は、光学切片のための装備します

ホールマウントゼブラフィッシュ胚の繊毛器官におけるリン酸化エピトープを免疫染色

幼生ゼブラフィッシュ性腺組織の解剖

ゼブラフィッシュ腫瘍異種移植片における宿主血管新生応答の生体内イメージングと定量

全体のマウント蛍光In Situハイブリダイゼーション、免疫組織の結合されたプロトコルを介してゼブラフィッシュにおける Multiciliated の細胞を可視化します。

骨組織の再生にプレドニゾロンの効果を研究する大人のゼブラフィッシュ傷害モデル

ブライトフィールド顕微鏡を用いた幼虫ゼブラフィッシュの肝臓サイズの定量化

アダルトゼブラフィッシュ腎臓の解剖

魚の目を通して糖尿病の勉強: 大人 tg(fli:EGFP) ゼブラフィッシュ網膜血管系の解析、可視化、レーザーマイクロダイ セクション

ホールマウントによるゼブラフィッシュ胚標本ステンドの観察および分析のためのフラットマウントの準備

魚の目を通して糖尿病の勉強: 大人 tg(fli:EGFP) ゼブラフィッシュ網膜血管系の解析、可視化、レーザーマイクロダイセクション