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。

パート1：胚を修正<ol><li>氷冷DEPC - PBSで解剖皿を埋める。氷上に置きます。ピンセットで殻をタップして卵を開いて、シェルの部分を削除してください。 </li><li>鉗子によるアルブミン厚い取り外します。その胚が上向きに向くように粗い鉗子で卵黄嚢を傾けて。 </li><li>はさみを使って、胚の周りに卵黄嚢の正方形をカット。 </li><li>スプーンを使用して、氷冷DEPC - PBSで卵黄と場所から胚を削除します。 </li><li>顕微鏡下で、膜と卵黄を削除し、固定の皿に移す。 </li><li>突き止める、DEPC - PBSを除去。 DEPC - PBSで4％PFAで置き換えると4でO / Nを修正℃に</li><li>固定液取り外します。氷冷DEPC - PBSに交換してください。平滑末端マイクロキャピラリーニードルまたはマイクロダイセクションのナイフを使用して、プローブのトラッピングを防ぐために、神経系および心腔を穿孔する。 </li><li> 0.1％のTween - 20（PBT）と2倍の10分DEPC - PBSを洗ってください。 25％、50％、PBTで75％メタノールで各10分を脱水する。 100％メタノールで二回胚を脱水する。メタノールで-20℃で保存。 </li></ol>パート2：プレハイブリダイゼーション<ol><li> PBTで75％、50％、25％メタノールでそれぞれの胚を10分を再水和。 PBTの2倍の10分を洗ってください。 </li><li>一方、氷冷固定液（PBTで4％パラホルムアルデヒド）を準備する。プロテイナーゼK溶液（; [か5 g / mlの]最後のPBTの3ミリリットル/バイアル）とPBTを交換してください。全体バイアルを静かにバイアルを圧延してプロテイナーゼK溶液にさらされていることを確認します。露光時間のトラブルシューティングを参照してください。 </li><li> RNaseフリーパスツールピペット削除プロテイナーゼKを使用し、固定液（2 ml）を加える。すぐに正確に20分、氷上に置きます。 </li><li>すべてのRTの洗浄はnutator上で実行されます。 2倍のPBTで10分、および50％のハイブリダイゼーションバッファーを含むPBTの1X 10mnを洗ってください。 </li><li>ハイブリダイゼーションバッファーで1倍を洗ってください。 65℃でインキュベート4〜5時間、2 mlのハイブリダイゼーションバッファー中のC。 </li></ol>パート3：プローブとハイブリダイズ胚<ol><li> DIGとFITC結合プローブの合成は、付録Aで説明されています。強い信号を与えると予想プローブは、FITC含有ヌクレオチドでsynhesizedされるべきである;弱いプローブは、DIG -を含むヌクレオチドとを用いて合成する必要があります。 </li><li> 2mlのバイアルを使用してハイブリダイゼーションバッファーにPrewarmプローブ（500 ng / mlの各）。速やかにプローブ溶液とハイブリダイゼーションバッファーを交換してください。胚は乾燥させないでください。 65℃16時間インキュベート℃に</li><li>ハイブリダイゼーション緩衝液、2回洗浄、30分65℃の各℃でプローブを交換してください65でハイブリダイゼーション緩衝液/ MABT（1 / 1 V / V /）で15分を洗う℃に</li></ol>パート4：DIG抗体のインキュベーションと呈色反応<ol><li> MABTで2 ×分を洗浄する。 2％BBR / MABTで1時間を洗います。 2％で5時間を洗うBBR/20％HISS / MABT（ブロッキングバッファー）。ブロッキングバッファーで希釈1:2000 DIG抗体でインキュベートし、4℃でO / N ° nutatorでC。 </li><li> MABTの3倍の10分ごと、および3倍の1時間MABT内の各を洗う。 </li><li> NTMTの2倍の20分を洗ってください。 10 mlのNTMTに200ulのNBT / BCIP基質を追加。すぐにバイアルからNTMTを削除し、1〜2ミリリットルNBT / BCIPバッファーと交換してください。暗闇の中で場所。その後20分後、および20分間隔で呈色反応を監視する。 </li><li>呈色反応（青色表示される）に続いて、10分のためにPBSで洗浄し、PBSで満たした固定皿に突き止める、そしてPBS中4％PFAでソリューションを交換してください。 4℃でO / N℃を修正</li><li>新しいシンチレーションバイアルに移す。 2 × 10分（1％のTween - 20を含むPBS）PBSTで洗浄する。 MABT 2倍10分で洗浄する。 </li><li> 63℃でMABT 30分でインキュベート</li></ol>パート5：FITC抗体のインキュベーションと呈色反応<ol><li> MABTの2倍10分、2％BBR / MABT、ブロッキング緩衝液で5時間の1時間を洗う</li><li>アルカリホスファターゼ結合抗FITC -抗体（1:500）で4 O / N ° Cでインキュベートする</li><li> MABTの3倍の10分ごと、および3倍の1時間MABT内の各を洗う。 </li><li> 0.1％のTween - 20（TBST）でTBS pHは8.45で2倍の20分で洗浄する。 </li><li>キットに含まれる試薬1,2および3を使用してTBSTでベクトルレッドワーキング溶液（5 ml）を準備する。すぐにバイアルからTBSTを削除し、ベクトルレッドバッファーと交換してください。暗闇の中で場所。 40分後、およびその後30分間隔で呈色反応を監視する。 </li><li>以下の呈色反応（赤色表示されているはずです）、PBSに転送胚。 </li></ol>第6部：固定および処理<ol><li> PBSを含む固定の皿に移し、そして4℃、RTまたはO / Nで約20分を4％PFAで固定し℃の</li><li> PBS、2倍の10分で洗浄する。写真撮影のために処理するために、PBS中の20％グリセロール（これは、胚を半透明になる）に移す。チャンバーを作成するには、PVCテープの2ストリップをカット、スライド上に重ね合わせる。ブレードを使用して中央に正方形をカット。鉗子を使って正方形を削除します。 </li><li>ワックスのセクショニングのために処理するために、メタノールの段階的な一連の（25％、50％、75％、PBSで100％、10分ごと）で水分を取り除き、PBS、洗浄2倍の10分に戻って転送する。 </li><li>プロパノール、3MNと交換してください。 Histoclear（2 × 1時間）に続いて1,2,3,4 - テトラヒドロナフタレン、約20分で交換してください。 1時間histoclear /ワックス1 / 1（v / v）を60 ° Cで置き換えます。ワックスを使用してプロセスを置き換える組織学用。 </li></ol>パート7：ソリューション -20ハイブリダイゼーション緩衝液、店舗°ファルコンでC、50 mlの25 mlのホルムアミド、3.25ミリリットル（20xSSC）、0.5ミリリットル（0.5M EDTA）、1ミリリットル（10％のTween - 20）、2.5ミリリットル（1％SDS） 、125、UL（20mg/ml tRNA）が、。100ul（50 mg / mlのヘパリン）MABTは、100mMのマレイン酸を用意し、pH7.5にNaOHペレットで。 150mmのNaClおよび0.1％Tween - 20を追加。 NTMT（使用直前に作られた）を、50ml：（5M NaCl）を1ミリリットル、2.5ミリリットル（2Mトリス- HCl pH9.5）、1.25ミリリットル（2MのMgCl 2）、5ミリリットル（10％のTween - 20）。 TBST：0.1Mトリス- HCl、0.15MのNaCl、pHは8.45、0.1％のTween - 20。 パート8：トラブルシューティング<table border="1"><tbody><tr><th>問題が</th><th>原因と</th><th>救済策</th></tr><tr><td valign="top">胚は丸くなっている</td><td valign="top">不足して脱水/再水和</td><td valign="top">連続して脱水/再水和ステップの間に10分間隔を維持する</td></tr><tr><td valign="top">プローブの信号はありません</td><td valign="top">プローブは、バイアルの劣化したRNaseのコンタミです。 </td><td valign="top">全体バイアルは、手順2でプロティナーゼK溶液にさらされていることを確認して1％アガロースゲル上でプローブを実行する</td></tr><tr><td valign="top">プローブは、心臓のラベルの空洞神経管です。 </td><td valign="top">プローブがトラップされ</td><td valign="top">ステップ2では、すべてのキャビティに穴が使用して顕微解剖ナイフまたはマイクロキャピラリーていることを確認してください</td></tr><tr><td valign="top">胚は、以下のハイブリダイゼーション（ステップ3）崩壊</td><td valign="top">プロテイナーゼKのステップでは、長すぎるハイブリダイゼーションの温度が高すぎるまま</td><td valign="top">プロテイナーゼKは長い1mn以上のままにしないでください（ステージ3 +と4）、2 MN（ステージ5〜7）、3分（ステージ8-10）、4 mnは（ステージ11-13）より高いT Oでハイブリダイズしないでください。よりも65 ° Cの</td></tr></tbody></table>パート9：代表的な結果代表的な胚を以下に示します：（A）胚（ステージHH7は）DIG -ソックスプローブ（青）とFITC -デルタ- 1プローブ（赤）で標識されている。 （B）胚（ステージHH8は）DIG - Pax6プローブ（青）とFITC - Shhのプローブ（赤）で標識されている。 NF、神経襞、ANP、前方神経板、PSM、前体節中胚葉、nは、脊索、NT、神経管）。博士の研究室からのプローブの贈り物。 C. Tabin、M.グールディング、D.エンリケ、そしてJ.ブリスコー。 <img src="/files/ftp_upload/old/904_2008_10_20_1/904_Figure-01.png" alt="" width="303" height="300" />

<ol>
	<li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., 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=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. 406, 74-78 (2000).</li><li>Rodr&#237;guez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A., Izpis&#250;a Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+Cer-like+protein+Caronte+mediates+the+establishment+of+embryonic+left-right+asymmetry.">The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.</a> Nature. 401, 243-251 (1999).</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><li>Kawakami, M., Nakanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+endogenous+PKA+inhibitor,+PKIalpha,+in+organizing+left-right+axis+formation.">The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation.</a> Development. 128, 2509-2515 (2001).</li><li>Basch, M. L., Bronner-Fraser, M., Garcia-Castro, M. I. <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+the+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of the neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li></ol>

The authors have nothing to disclose.

in situハイブリダイゼーション法2色全体のマウントは、関心の1つまたは2つの遺伝子を用いて、遺伝子発現の空間的および時間的パターンの両方を決定するために使用されます。アプリケーションでは、新規遺伝子の遺伝子発現パターンの評価（例えば1,2、だけでなく、遺伝子発現、次の侮辱の変化（胚操作3、ビーズ4、RNAまたはDNAのエレクトロポレーション5構築）が含まれ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<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>Animal</td>
<td>Charles River </td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Hybaid</td>
<td>Micro-4</td>
<td>&nbsp;</td>
<tr>
<td>Adams Nutator orbital mixer</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-062</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>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Spoon</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10370-18</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Keep pins together using a magnetic stirrer;</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>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379-100ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2308-5MG</td>
<td>Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>Formamide, deionized</td>
<td>Reagent</td>
<td>Ambion</td>
<td>9342</td>
<td>&nbsp;</td>
<tr>
<td>Yeast tRNA-25 mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15401-011</td>
<td>Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C</td>
</tr>
<tr>
<td>Heparin Sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4784-250MG</td>
<td>Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>SSC x20 pH5.5</td>
<td>Reagent</td>
<td>Fisher</td>
<td>PR-V4261</td>
<td>&nbsp;</td>
<tr>
<td>EDTA (0.5M)</td>
<td>PR-V4231</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP166-100</td>
<td>&nbsp;</td>
<tr>
<td>Maleic acid</td>
<td>Reagent</td>
<td>Fluka</td>
<td>63189</td>
<td>&nbsp;</td>
<tr>
<td>Boehringer Blocking Reagent</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11096176001</td>
<td>Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.</td>
</tr>
<tr>
<td>Heat inactivated sheep serum</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>013-000-121</td>
<td>Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>NBT/BCIP stock solution</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11681451001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>2M Tris</td>
<td>BP1759-500</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vector Red Alkaline Phosphatase Substrate Kit</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>SK-5100</td>
<td>&nbsp;</td>
<tr>
<td>1,2,3,4-tetrahydronaphthalene</td>
<td>Reagent</td>
<td>Sigma</td>
<td>429325-100ML</td>
<td>Under hood</td>
</tr>		
</tbody>
</table>
</div>

double whole mount in situ hybridization of early chick embryos

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、体節、心臓原基の形成における遺伝子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、in situハイブリダイゼーションの2色全体のマウントで別の手順を示し、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、胚は、プレハイブリダイゼーションのために処理されます。胚は、2つの異なるプローブを、DIGに結​​合された1つ、およびFITCに結合された1つでハイブリダイズさせる。一晩ハイブリダイゼーション後、胚はDIG結合抗体とインキュベートされる。 DIG基板用色反応を行い、関心領域は青色表示されます。胚は、その後、FITC結合抗体とインキュベートされる。胚は、FITCによる呈色反応のために処理、および関心領域は、赤色表示されます。最後に、胚はphtographとセクショニングの固定され、処理されます。トラブルシューティングガイドも表示されます。

Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Double Whole Mount in situ Hybridization of Early Chick Embryos

このビデオでは、in situハイブリダイゼーション2色全体のマウント、2つの異なる遺伝子の空間的および時間的発現パターンは、若いニワトリ胚で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、Davidウィルキンソン、ドミンゴスエンリケ、フィルインガムとデビッドイシュ- Horowiczによって導入された。

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

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

double-whole-mount-in-situ-hybridization-of-early-chick-embryos

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JoVE Journal

Biology

12.3K ビュー.  Institute of Biosciences and Technology - Texas A&M Health Science Center. このビデオでは、in situハイブリダイゼーション2色全体のマウント、2つの異なる遺伝子の空間的および時間的発現パターンは、若いニワトリ胚で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、Davidウィルキンソン、ドミンゴスエンリケ、フィルインガムとデビッドイシュ- Horowiczによって導入された。

ビデオ: Double Whole Mount in situ Hybridization of Early Chick Embryos

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

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

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 ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

鶏胚における単一細胞のトランスフェクション

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

初期のニワトリ胚の形態形成の組織の変形を追跡

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解像度全マウント<em&gt;その場で</em遺伝子発現と機能を研究するためにゼブラフィッシュ胚内&gt;ハイブリダイゼーション

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...