NIHグラントR01EY016099と盲目のキャリア開発賞を防止する研究：この研究は国立衛生研究所（NIH）コアグラント（5P30EY008098）と、以下の資金XWのによってサポートされていました。我々は、共焦点イメージングにおける援助のためにキラのラスロップに感謝。

1日目。準備 1。魚の交配を設定する夕方にはゼブラフィッシュのペア十字架を設定します。希望の時間の前にランダムな交配を防ぐために、交配のカセットで、女性と男性の魚を分離する仕切りを使用してください。 GFP -発現するトランスジェニックフィッシュとドナー割球のソースとして使用されます。 pt104トランスジェニックライン（ゾウら 、2008）、ここで使用される、EF1プロモーターの制御下で普遍的にGFPを発現する。 GFPの発現は、ライブイメージングによるドナー細胞の可視化を可能にする。適切な蛍光タンパク質を発現させる他のトランスジェニック魚の行は、特定のニーズに合わせて使用​​することができます。 2。保持する胚のためのくさび形のアガロースの井戸を準備シャーレにE3卵水（ウェ、2007）で調製した溶融1％アガロースを、、注ぐ。アガロース溶液にくさび状突起を持ち、アガロース溶液が完全に固化させプラスチック金型をフロートさせます。慎重に金型を取り外し、E3水でアガロースのベッドを浸す。 3。移植針を準備ガラスキャピラリーピペット（世界の精密機器を）引いて針を作るために11.5電力設定で垂直ニードルプラー（デビッドKopfは楽器を）使用してください。良い針先は長いシャフトでテーパーすべき。内径40 -45μmの傾斜開口部が得られるまでマイクログラインダー（Marishige）と45度の角度で針先を挽く（図1A）。針先をクリーンアップするには、吸うと表示されているすべての残骸が削除されるまで10％フッ化水素酸溶液を解放して注射器にホースを接続し、針の平滑末端にホースを取り付け、と。次に、100％エタノール、続いて蒸留水でのヒントを洗う。すべてのエタノールが蒸発するまで針を空気乾燥させる。残留エタノールは、高温の研磨工程の間に針を台無しにする：ヒントが完全に乾いていることを確認することが重要です。針の先端のギザギザを柔らかくし、研磨する、マイクロ捏造（Marishige）のフィラメントによって発生する熱と針の先端を焼く。針の先端が研磨中にフィラメントを接触させてはいけません。フィラメントと針の先端との間のフィラメントとの距離も通過する電気の量は、所望の加熱条件を達成するために調整することができます。温度が高すぎるすべきではない：これは、針の先端を溶融して変形します。粗いエッジが平滑化された後、慎重に針の先端に加熱されたフィラメントに触れ、その後穏やかに先端（図1B）を生成するために針から離れてフィラメントを引き出します。 、シャープな滑らかな、そしてきれいな針はそのままドナー割球を得るためと宿主胚を損傷しないために重要です。 4。胚位置決めプローブを準備滑らかに端を細いガラスプローブを得るためにガスの炎を介してガラスピペットを引き出します。このプローブは、移植に先立って適切な向きに胚を配置するために使用されます。 2日目。割球移植 1。収集とdechorionate胚翌日、チームメイトに魚を可能にするクロスカセットに仕切りを削除する。 E3水を満たしたシャーレに胚を置きます。その後、手動で微ピンセットで胚からchorionsを離れて引き裂くことによりdechorionate胚。慎重にアガロースウェルにdechorionated胚を転送するために曲がったシャフトをガラスピペットを使用してください。バーストに胚を引き起こす空気と胚、任意の接触を避ける。胚は28.5で開発℃で3時間後に受精、またはHPFになるまでアガロース井戸でCをしましょう​​。 2。移植装置を設定する胚が発達しながら、鉱物油で満たされたマイクロポンプシステムへの移植の針を接続することにより、移植装置を設定します。システムがリークされず、空気の泡がシステムの中に閉じ込めされていないことを確認します。先端開口部のスペースを1μl - バック0.5を除いて、ミネラルオイルを全体移植針を埋める。 3。割球の移植割球を移植する、上方3 - 4 - HPFの胚の割球側をオリエンテーションするガラスの位置決めプローブを使用してください。その後、そっとドナー胚に移植針を挿入し、ゆっくりと針に5〜20割球を吸い上げる。宿主胚に割球を充填した針を挿入し、徐々に割球を離します。彼らの子孫が後の発達段階でローカライズどこ割球がリリースされるサイトが影響します。網膜と脳の発達、動物極でのリリースの細胞を分析する。移植時に、針の先端が空気に接触しないことを確認してください。機械的損傷を最小限に抑えるために、宿主胚は、次の日まで、28.5 ° Cでアガロースのウェルに残されるものとする。次に、t各ウェル内の1つの胚を、1％アガロース、0.5 mlのプレコートした24ウェルプレートにモザイク胚のケース温度。 E3卵の水1 mlを各ウェルに追加されます。細菌感染を防ぐために、E3卵の水に100倍のペニシリンとストレプトマイシンの10μlを加える。 3日目。埋め込みとモザイク胚のライブイメージング 1。埋め込み胚 E3卵の水に1.5％低融点アガロース（LMP）を準備し、30℃の水浴でソリューションを維持。厚さ0.17ミリメートル（世界の精密機器）のグラスの底部とFluoroDish組織培養皿にモザイク胚を移す。ちょうどE3の最小限の量でカバー胚を残すために余分なE3の水分を取り除く。胚30〜50μlを溶かした1.5​​％LMPを追加。アガロース溶液が固化する前に、可能な限り迅速にガラスの底の近くに眼や脳を配置します。解決策が冷却した後、さらに場所の胚を確保するために追加のアガロース溶液を加える。その後、2〜3ミリリットルE3の卵の水で固化したアガロースブ​​ロックを浸す。 2。共焦点像共焦点顕微鏡の場合は、大規模な焦点深度の水浸反転目標（オリンパス、PlanApo、40 X/0.90 WLSM、日本）が使用されます。水の滴は、客観的に追加して、客観的にFluoroDish組織培養皿を置いています。画像の開発に、胚の写真を撮り、5分ごとにタイムラプスイメージングを行う。実験の性質に応じて、ショットの間の時間間隔を調整する必要があります。 代表的な結果図2は、代表的な24 - HPFモザイクのゼブラフィッシュを示しています。 GFPの発現は、緑のドナー細胞を強調。網膜神経上皮細胞は網膜の壁の厚さ全体に及ぶ。 映画は24 HPFでのホストの脳におけるドナー細胞の動きを示しています。時間をかけて細胞の形態変化に注目してください。 <img src="/files/ftp_upload/1924/1924fig1.jpg" alt="図1" /> 図1。移植の針先の形状。 A.は、針の先端の斜めの開口部は、フッ酸洗浄後の残骸の自由だ。 B.先端が針の先端に生成されました。 <img src="/files/ftp_upload/1924/1924fig2.jpg" alt="図2" /> 図2。24 HPFにおけるキメラゼブラフィッシュの胚におけるGFP発現ドナー細胞の可視化。左側のパネルには、脳と網膜におけるGFPのシグナルを示しています。中央のパネルには胚の明視野像である。右側のパネルには、マージされた画像を示しています。 映画は1。GFP陽性ドナー細胞のイメージングが開発中の脳の神経上皮細胞の形態には大きな変更を示したライブ。映画の個々のフレームは、24及び25 HPFの間に5 ​​分ごとに収集された。 <a href="http://www.jove.com/files/ftp_upload/1924/1924_Wei_Movie1.wmv">映画を見るにはここをクリック</a> 。

<ol>
	<li>Zou, J., Lathrop, K., Sun, M., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+RPE+maintained+by+Nok+is+essential+for+retinal+epithelial+polarity+and+cellular+patterning+in+zebrafish.">Intact RPE maintained by Nok is essential for retinal epithelial polarity and cellular patterning in zebrafish.</a> Journal of Neuroscience. 28 (50), 13684-13695 (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=A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> THE ZEBRAFISH BOOK. , (2007).</li><li>Ho, R. K., Kane, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+action+of+zebrafish+spt-1+mutation+in+specific+mesodermal+precursors.">Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors.</a> Nature. 348, 728-730 (1990).</li><li>Catalano, A., Raymond, P., Goldman, D., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dou+yan+Mutation+Causes+Patterning+Defects+and+Extensive+Cell+Death+in+the+Retina.">Zebrafish dou yan Mutation Causes Patterning Defects and Extensive Cell Death in the Retina.</a> Developmental Dynamics. 236 (5), 1295-1236 (2007).</li></ol>

このビデオでは、我々はゼブラフィッシュの網膜や脳の発達を分析する割球移植する方法を示しています。このプロトコルはまた、単に、ホスト胚の対応する場所にドナー割球を放出することによって、他の組織や器官の発達を分析するために使用することができます。 GFP分子による標識は、蛍光色素標識デキストラン（ホーとケイン1990）で標識よりクリーンなバックグラウンドをもたら?...

E3の卵の水： <ul><li> 5mMのNaClを</li><li> 0.17 mMのKClを</li><li> 0.33 mMのCaCl 2を </li><li> 0.33 mMのMgSO 4を </li><li> 0.1％メチレンブルー</li></ul>

transplantation of gfp-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos

細胞は、胚形成の間にその場所とプロパティで広く変わる。これらの変化は、細胞とその環境との相互作用によって調節される。異なる遺伝的背景のセルで構成されているキメラ胚は、、、目的の遺伝子により媒介される細胞間相互作用を研究するための優れたツールです。初期の発達段階におけるゼブラフィッシュの胚の透明性は、細胞レベルでの組織や臓器の形態形成の直接可視化を可能にします。ここで、我々は、ゼブラフィッシュ胚におけるキメラ網膜と脳を生成するために、ドナー細胞のライブイメージングを実行するプロトコルを示しています。プロトコルは、移植針、GFP陰性のホストへのGFP発現ドナー割球の移植、およびライブ共焦点顕微鏡下でドナー細胞の挙動の試験の準備をカバーしています。若干の修正で、このプロトコルはまた、ゼブラフィッシュの他の組織や臓器の胚発生を研究するために使用することができます。ラベルのドナー細胞にGFPを使用する利点についても説明します。

Watch this Scientific Journal Video about Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos at JoVE.com

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

我々は、胚発生時のライブイメージング細胞挙動のためのキメラゼブラフィッシュの胚を生成するプロトコルを示しています。

キメラゼブラフィッシュ胚における網膜と脳の開発のライブイメージングのためのGFP発現割球の移植

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

transplantation-gfp-expressing-blastomeres-for-live-imaging-retinal

Research

JoVE Journal

Biology

10.6K ビュー.  University of Pittsburgh. 我々は、胚発生時のライブイメージング細胞挙動のためのキメラゼブラフィッシュの胚を生成するプロトコルを示しています。

ビデオ: Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植によるキメラゼブラフィッシュ胚の生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

細胞運動やゼブラフィッシュ胚における個々の神経細胞と神経堤細胞のアクチン細胞骨格のライブイメージング

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ライブゼブラフィッシュ胚における二光子ベースの光活性化

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

キメラの生成のためのメダカ胚および細胞移植のDechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

代謝標識とBioorthogonalクリックケミストリーによるゼブラフィッシュ胚でのイメージンググリカン

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.