（J.ドッド、TM JessellとA.川上）によって開発されたモノクローナル抗体は（15.3B9、PAX7）生物科学科、アイオワ大学でNICHDと維持の後援の下に開発された発達研究ハイブリドーマバンクから入手した、アイオワ州。 DPは、薬物乱用の国民の協会からルースキルシュシュタイン賞1F32 DA021977 - 01A1の受信者です。この作品は、RHFへマーガレットM. Alkek財団によってサポートされていました。

I.の概要図： このビデオでは、ニワトリ胚での全体のマウント免疫組織化学での別の手順を示しています。最初に、胚はPFAで固定されています[IHC1]。次に、内因性ペルオキシダーゼ活性をクエンチされる[IHC2]。胚は、その後一次抗体[IHC3]でインキュベートする。数回の洗浄後、胚を二次抗体で[IHC4]をインキュベートされ、色の反応は、DAB [IHC5]と抗体染色を用いて明らかにされるオレンジ色の[IHC6]が表示されます。 <img src="/files/ftp_upload/old/956_2009_1_26_3/figure 1.png" alt="図1" width="600" height="573" />パート1：胚を修正<ol><li>ニワトリ胚での全体のマウント免疫組織化学を実行するには、最初にピンセットで殻をタップし、シェルの部分を削除して、卵を開きます。 </li><li>鉗子と厚いアルブミンを削除して、胚が上向きに向くように粗い鉗子で卵黄嚢を傾けます。 </li><li>細かいハサミを使用して、胚の周りに卵黄嚢の正方形をカット。スプーンで卵黄から胚を取り出し、そしてPBSを含む皿の場所。 </li><li>解剖顕微鏡下で、慎重に膜と卵黄およびPBSを含む新鮮な皿に胚移植を削除します。 </li><li>鉗子や昆虫ピンで胚を突き止める、およびPBSを吸引除去する。 PBS中4％PFAでこれを交換し、胚は、室温で1時間を修正することができます。 </li></ol>パート2：抗体のステップの準備胚<ol><li>潜在的な微生物の汚染を最小限に抑えるために、すべて以下の手順でのddH 2 Oを使用してください。 </li><li>胚を固定された後、固定液を吸引除去し、化学廃棄物として適切に処分する。 PBSで皿を埋める。 </li><li>次に、顕微解剖ナイフを用いて、胚体外膜を除去するために胚の周りの角をカット。ピンセットで虫ピンを取り外します。 </li><li>ピンが削除された後、PBT（PBS pH7.4の、0.5％トリトンX）とを含むシンチレーションバイアルへ胚を転送するために平滑末端パスツールピペットを使用してください。 </li><li> 、PBTで10分間ずつ3回の胚を洗浄してください。 </li><li>シンチレーションバイアルからPBTを削除し、PBTxは潜在的な内因性ペルオキシダーゼを不活性化するために0.3％H 2 O 2を含むと交換してください。 </li><li> nutator上で室温で2時間インキュベートする。 </li><li>その後、PBT、10分ごとに3回、30分ごとに3回で胚を洗浄してください。 </li></ol>パート3：抗体のインキュベーション<ol><li>抗体で胚を染色するために、ブロッキング緩衝液または1％BSA / 1％NGS / PBT（二次抗体がヤギで飼育されているため、我々はNGSを使用）で1時間胚を洗浄することにより開始します。 RTで1時間nutatorでインキュベートする。 </li><li> 4℃で2日間nutator上でCは、この溶液中でブロッキング緩衝液、及びインキュベート胚で一次抗体1:1に希釈する。一次抗体の希釈率は、一次抗体の選択に依存する。ここで、我々は、上澄みとして提供されている発達研究ハイブリドーマバンクからの抗体を使用してください。我々は、ブロッキング緩衝液で1:1に希釈する。 </li><li>次に、PBTで3 1時間の洗浄に続いてPBT 3位10分間の洗浄を、実行してください。 </li><li>洗浄後、ブロッキング緩衝液で二次抗体を1:2500に希釈する（この場合ペルオキシダーゼ共役ヤギ抗マウスIgG（H + L）、そしてこの溶液中でインキュベート胚の4でO / N ° nutatorでC。 </li><li> PBTの3 1時間の洗浄に続いてPBT 3位10分間の洗浄を、実行します。 </li></ol>パート3：色反応<ol><li>ステンド胚の呈色反応を開発するには、まずトリス緩衝液（100mMのトリスHCl、pH7.4）中2 20分間の洗浄を行います。 </li><li>一方、ヒュームフードの下で500μg/mlのトリス緩衝液中で（3,3&#39; - ジアミノベンジジン四塩酸塩）DAB基質を溶解し、氷の上に、暗闇の中で解決策を保つ。 </li><li>バイアルを含む胚から最後のTrisバッファー洗浄を削除し、ステップ3.2から5ミリリットルDAB溶液を交換してください。約20分のためのnutatorで暗所に保管してください。 DABを除染するために10％漂白剤溶液の入ったバケツにエッペンドルフ先端処分する。 </li><li>一方、0.3％の株式氷上のdH 2 O中のH 2 O 2を準備。 </li><li> DABで胚を含むバイアルに50μlのストックH 2 O 2を追加。暗所で保管してください。 1〜2分後、顕微鏡下で反応を監視する。 </li></ol>パート4：写真撮影​​および組織学のための胚の処理<ol><li>呈色反応が完了すると、漂白剤をバケツにDAB処分する。 5 mlの水道水に交換してください。 </li><li> PBSで2 10分間の洗浄を行います。 </li><li>写真とワックスのセクショニングのために処理するには、10分ごとに（25％、50％、75％、100％）エタノール系列で脱水。 </li><li> 0.5でエタノールを交換してください - 1ミリリットルシダーウッドオイルを（これは、胚を半透明になります）。杉の木の油で写真撮影のためのプロセス。 </li><li>以下の写真は、バイアルに胚を戻すと、100％エタノールで杉の木のオイルを交換してください。エタノールの手順を繰り返します。 </li><li>メールを交換してください100％エタノールに5％ファストグリーンFCFとthanol、と（このステップは、組織学のための胚を閲覧できるようになります）3分間インキュベートする。組織学のための100％エタノールとプロセスとファストグリーンFCFのソリューションを交換してください。 </li></ol>代表的な結果： <img src="/files/ftp_upload/old/956_2009_1_26_2/figure2.png" alt="図2" width="600" height="498" />以下に示す例では、胚が段階HH​​ 10（）、12（B）と11（C）で解剖され、胚は、新興神経crestasにPAX 7の発現を見せるだけでなく、体節と神経管（A、B）の; （C）においては、脊索は15.3B9（はA - 1）（発達研究ハイブリドーマバンクから提供された抗体）で標識されている。

<ol>
	<li>Yamada, T., Placzek, M., Tanaka, H., Dodd, J., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+developing+nervous+system:+Polarizing+activity+of+the+floor+plate+and+notochord.">Control of cell pattern in the developing nervous system: Polarizing activity of the floor plate and notochord.</a> Cell. 64, 635-647 (1991).</li><li>Streit, A., Stern, C. D., Thery, C., Ireland, G. W., Aparicio, S., Sharpe, M. J., Gherardi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+HGF/SF+in+neural+induction+and+its+expression+in+Hensen's+node+during+gastrulation.">A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation.</a> Development. 121, 813-824 (1995).</li><li>Basch, M., Bronner-Fraser, M., Garcia-Castro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li></ol>

このビデオでは、若いニワトリ胚のホールマウントの抗体染色を行うことで様々なステップを示しています。このプロトコルは、本質的に空間的および時間的ひよこ2,3における新規抗体の特徴だけでなく、侮辱4次胚の奇形を決定するために、既知の抗原マーカーの使用のために使用されます。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH4 (16 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Other</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td> Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to cut embryo from surrounding membranes following fixation</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>30% H2O2</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1009</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3803</td>
<td>&nbsp;</td>
<tr>
<td>NGS</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>005-000-121</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibodies</td>
<td>Reagent</td>
<td>Developmental studies Hybridoma Bank</td>
<td>4G11, 15.3B9, PAX7</td>
<td>For these antibodies, investgators used mouse donnors</td>
</tr>
<tr>
<td>Peroxidase conjugated-Goat Anti-Mouse IgG (H+L) </td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>115-035-003</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride</td>
<td>Reagent</td>
<td>Pierce</td>
<td>34001</td>
<td>Store at -20; Allow bottle to warm to RT before use.</td>
</tr>
<tr>
<td>Cedar wood oil</td>
<td>Reagent</td>
<td>Sigma</td>
<td>W522503</td>
<td>&nbsp;</td>
<tr>
<td>Fast green FCF</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20 </td>
<td>&nbsp;</td>		</tbody>
		</table>
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method for whole mount antibody staining in chick

ニワトリ胚は、初期胚発生の研究に貴重なツールです。その透明性、アクセス性と操作の容易さ、それを脳、神経管や体節の開発に抗体発現を研究するための理想的なツールとなっています。このビデオでは、HRP標識二次抗体を用いたホールマウントの抗体染色で別の手順を示し、まず、胚は卵から解剖し、パラホルムアルデヒドで固定されています。第二に、内因性ペルオキシダーゼの不活化であり、胚は、その後一次抗体にさらされています。数回の洗浄後、胚は、HRPにコンジュゲートした二次抗体とインキュベートされる。ペルオキシダーゼ活性は、ジアミノベンジジン基質との反応を用いて明らかにされる。最後に、胚を固定して、写真とセクショニングのために処理されます。蛍光抗体の使用に比べてこの方法の利点は、胚は、このような断面で抗原部位の調査を可能にする、ワックスのセクショニングのために処理できることです。このメソッドは当​​初、ジェーンドッドとトムJessellによって導入された<sup&gt; 1</sup&gt;。

Watch this Scientific Journal Video about Method for Whole Mount Antibody Staining in Chick at JoVE.com

Method for Whole Mount Antibody Staining in Chick

このビデオでは、全体のマウント免疫組織化学、抗原の空間的および時間的発現パターンは、若いニワトリ胚における可視化することができる方法を示しています。このメソッドは当​​初、ジェーンドッドとトムJessellによって導入されました。

チックの全マウントの抗体染色のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

method-for-whole-mount-antibody-staining-in-chick

Research

JoVE Journal

Biology

15.6K ビュー.  Texas A&M University (TAMU). このビデオでは、全体のマウント免疫組織化学、抗原の空間的および時間的発現パターンは、若いニワトリ胚における可視化することができる方法を示しています。このメソッドは当​​初、ジェーンドッドとトムJessellによって導入されました。

ビデオ: Method for Whole Mount Antibody Staining in Chick

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

ゼブラフィッシュの全マウント高分解能二重蛍光上皮内ハイブリダイゼーション

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

全マウントを使用するその場で</em分子とOrganismal生物学をリンクする>ハイブリダイゼーション

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全体のマウント<em&gt;上皮内</emE11.5マウス胚へのE8.5&gt;ハイブリダイゼーション

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

ホールマウントRNAを蛍光<em&gt;その場で</emの&gt;ハイブリダイゼーション<em&gt;ショウジョウバエ</em&gt;胚

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNAを<em&gt;その場</emホールマウント胚および細胞組織学で&gt;ハイブリダイゼーションは、海洋軟骨魚類に適合した

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体染色中<em&gt; C.虫</em&gt;「フリーズクラッキング」使用

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

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 ...