The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

FLC40-14：動物で行わ全ての実験はCICUAL-プロトコル番号の下、メキシコ自治大学（UNAM）の実験動物管理使用委員会によって承認されました。 （CICUAL：「ComitéInstitucionalパラエルCuidado yの嘘・デ・ロス・Animales・デ・ラボラトリ・デル・研究所・デ・FisiologìaCELULAR、ナショナル大学自治・デ・メキシコ」）。 胚のタンパク質抽出物の調製<ol><li>各条件は、例えば72時間後に受精（HPF）のために、所望の段階で（モルファント対野生型）を比較するための200の胚 - 100を収集します。 </li><li>先の細いピンセットを使用して場所の魚の水を含むペトリ皿中の胚（ 表1参照）、手動dechorionate胚。それは、標的受容体を消化することができるように、この段階（またはプロトコルを通じて、他のプロテアーゼ）中にプロナーゼを使用しないでください。 </li><li> 1.5mlマイクロチューブに入れ胚と胚TWを洗います1×リン酸緩衝溶液（PBS）と氷（ 表1参照）。 </li><li> （ 表1を参照）deyolk用緩衝液500μlを追加します。 </li><li> （ - 40倍、約30）溶液中で胚を優しく上下にピペッティングにより卵黄のほとんどをリリース。 72 HPFと48 HPFの胚については、それぞれ、青と黄色のピペットチップを使用します。黄色のヒントが少し胚を壊さないようにカットする必要があるかもしれません。成功した卵黄の放出は、顕微鏡下で胚を観察することによって確認することができます。 </li><li> 15秒間600×gで遠心分離することによって胚を収集します。 </li><li>ピペットを使用して、慎重に上清を捨てます。 </li><li>洗浄胚は二回500μlの洗浄バッファーで穏やかに最低速でボルテックスすることによって（ 表1参照します）。 </li><li> 15秒間600×gで遠心分離することによって胚を収集します。 </li><li>ここから出発し、4°Cで手順を続行します。 </li><li>ピペットを使用して、慎重に上清を捨てます。 </li><li>溶解バッファー350μlの再懸濁胚（表1を参照）、プラスチック製乳棒を用いて均質化します。 </li><li> 4°Cで試験管ロッカーに溶解させた胚を攪拌しながら30分間インキュベート。 </li><li>遠心分離機は、不溶性の破片を除去するために15分間、11,000×gで胚を溶解しました。 </li><li>転送が新しいチューブにピペットを用いて上清をクリア。 </li><li> Bradfordタンパク質アッセイ4、または他の適切な手順により総タンパク質を決定します。 Bradfordアッセイを使用している場合、彼らは4標準タンパク質の過小評価を引き起こすように、溶解緩衝液中の界面活性剤の提示の等価な濃度の存在下で較正曲線を実行します。 </li></ol> 2.検出内因性受容体タンパク質の<ol><li> 親和性標識および免疫沈降（すべての手順4 O Cで） <ol><li> 1.5mlマイクロチューブに総胚タンパク質の500μgのバッファ1（ 表1を参照）を1μg/μlに希釈する- 400を置きます。 注：総胚タンパク質500μgのを得るためには、約100から200胚が最初に処理されなければなりません。 72 HPFの胚は、日常的にβグリカンの検出のために十分である総胚タンパク質、の〜5μgのをもたらします。 </li><li>試験管ロッカーに150 PMの最終濃度に達すると攪拌しながらインキュベートし、4℃で2時間標識TGF-β2株式の十分なボリュームを追加します。 AFLIPが開始される前にCheifetz ら 5によって説明したように、TGF-β2は、クロラミンT法によって125 Iで標識されなければなりません。 注意：使用して私は、リガンドを標識した125の処理中に露出を最小限にするためにシールド。 </li><li> 100希釈および（インキュベーションは、O / Nであってもよい）4℃でさらに2時間インキュベーションを継続：1に到達するために、目的の受容体に対する希釈していない抗体の十分なボリュームを追加します。これは、この研究で使用され、他の場所3を説明するが、ANの品質に応じて、抗血清＃31の最適希釈でありますtibody、より大きいまたはより小さい希釈が最適であってもよいです。 </li><li>適切な免疫グロブリン結合ビーズの50μlを添加（例えば、以前にTNTEで平衡化し、1再懸濁させたGタンパク質セファロース、：TNTEで元の体積の5）と4℃で試験管ロッカー上で撹拌しながら50分間インキュベートしますC. </li><li> 20秒間11,000×gで微量遠心によってビーズを回復。 </li><li>適切な放射性ゴミ容器に上清を捨てます。 </li><li> 20秒間11,000×gで毎回ボルテックスし、微量遠心することにより、（ 表1を参照）IP洗浄緩衝液1mlでIP-ビーズを3回洗浄します。 </li><li> IP洗浄緩衝液250μlの中に再懸濁したIP-ビーズ。 </li><li> （10 mg / mlとでDMSOに溶解したDSS、​​）ジスクシンイミジルスベレートの1.5μlを添加します。ちょうどこの段階でその使用前にDSS溶液を調製します。 </li><li>攪拌しながら4℃で15分間インキュベートします。 </li><li>架橋反応をクエンチするために、500μlのOを加えます十分トリス-Cl pH7.4の在庫を補充したF IP洗浄緩衝液は、25mMのトリス-Clに到達します。トリス中の遊離アミノ基が未反応のDSSをキャプチャします。 </li><li> 20秒IP-ビーズを収集し、上清を廃棄するための11,0​​00×gで遠心分離します。 </li><li>再懸濁IP-ビーズLaemmliバッファーを減らす30μlのインチ</li><li>沸騰サンプル94℃で5分間。 </li><li>必要に応じて、製造業者のプロトコルを使用して、ガンマカウンターでサンプルを分析します。 </li></ol></li><li> 試料分析 <ol><li>変性SDS-PAGEの対象サンプル。受容体の質量のための適切な割合でポリアクリルアミドを使用し、標準的な手順の下でゲルを実行します。 </li><li>固定液中でゲルを浸し、室温で30分間（ 表1参照）。 </li><li> 15分間蒸留水でゲルを洗浄してください。 </li><li>以前に水和ろ紙にゲルを置き、サランラップフィルムでカバーしています。 </li><li> 1時間80℃で乾燥ゲル。 </li><li>白ホスホイメージャSCREにゲルを公開RT O / Nでアン。 </li><li>製造業者のプロトコルを使用して、ホスホイメージャを用いて露光画面をスキャンします。 </li></ol></li></ol>

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The authors have nothing to disclose.

目的のタンパク質に対する特異的抗体を用いたウェスタンブロットの使用は、胚発生時にその発現7を研究するための貴重なツールです。しかし、高度にグリコシル化されたタンパク質のイムノブロットは、その非効率的な転送およびニトロセルロースまたはPVDF膜8,9に弱い結合に非常に成功していません。 プロテオグリカンは理由負に帯電してい...

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

<table><tbody><tr><td>Disuccinimidyl suberate (DSS)</td><td>ThermoFisher Scientific</td><td>21555</td><td></td></tr><tr><td>Protein G Sepharose 4 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0618-01</td><td></td></tr><tr><td>Gel Dryer Model 583&amp;nbsp;</td><td>BIO-RAD</td><td>1651745</td><td></td></tr><tr><td>Typhoon 9400</td><td>GE Healthcare Life Sciences</td><td>63-0055-78</td><td></td></tr><tr><td>Cobra II Auto gamma counter</td><td>Packard</td><td></td><td></td></tr><tr><td>Exposure Cassette</td><td>Molecular Dynamics</td><td>63-0035-44</td><td></td></tr><tr><td>NaCl</td><td>J.T. Baker</td><td>3624</td><td></td></tr><tr><td>KCl</td><td>J.T. Baker</td><td>3040</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3828</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3246</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;OH</td><td>J.T. Baker</td><td>9070</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;COOH</td><td>J.T. Baker</td><td>9508</td><td></td></tr><tr><td>HCHO</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>ED</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Crystal Sea Marine Mix</td><td>Marine Enterprises International</td><td>http://www.meisalt.com/Crystal-Sea-Marinemix</td><td></td></tr></tbody></table>

affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

図1は AFLIPで得られた代表的な結果を示しています。 125 I-リガンドから来レーン1の信号は、共有ゼブラフィッシュβグリカンコアタンパク質（BGコア、150 kDaのマーカー以下）またはグリコサミノグリカンのアタッチメント（GAG、スミアによってそのプロテオグリカンの形に加工されたBGコアのいずれかにリンクされています）170キロダルトンから?...

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

ゼブラフィッシュ胚からの内因性受容体の親和性ラベリング検出

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Research

JoVE Journal

Developmental Biology

5.6K ビュー.  Universidad Nacional Autónoma de México. A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

ビデオ: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

ゼブラフィッシュ後脳における標識およびイメージング細胞

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

神経科学

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 ...

発生生物学

<meta charset="utf8"></head><body>CRISPR/Cas9ノックインを用いたゼブラフィッシュ幼生の蛍光タンパク質標識

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>ゼブラフィッシュの低発現タンパク質を可視化するための蛍光標識

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

ゼブラフィッシュの低発現タンパク質を可視化するための蛍光標識

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

遺伝学

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

ゼブラフィッシュ胚と幼虫の全山免疫賦形化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

ゼブラフィッシュ放射状グリア前駆細胞の非対称分裂におけるNotch/Δエンドサイトーシスを追跡するための抗体取り込みアッセイ

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

レーザーUncagableフルオレセインデキストランでゼブラフィッシュ細胞の系統ラベリング

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

生物学

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

ゼブラフィッシュ胚における組織特異的蛍光の半自動イメージング

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

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 ...

ゼブラフィッシュ胚からの内因性受容体の親和性ラベリング検出

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ゼブラフィッシュ後脳における標識およびイメージング細胞

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。

ゼブラフィッシュの低発現タンパク質を可視化するための蛍光標識

ゼブラフィッシュの低発現タンパク質を可視化するための蛍光標識

ゼブラフィッシュの低発現タンパク質を可視化するための蛍光標識

ゼブラフィッシュ胚と幼虫の全山免疫賦形化学

ゼブラフィッシュ放射状グリア前駆細胞の非対称分裂におけるNotch/Δエンドサイトーシスを追跡するための抗体取り込みアッセイ

レーザーUncagableフルオレセインデキストランでゼブラフィッシュ細胞の系統ラベリング

ゼブラフィッシュ胚における組織特異的蛍光の半自動イメージング

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