我々はマウスの繁殖と介護の支援のために、実験室の管理のためのK.ギルに感謝。また、技術的なヘルプのための向山研究室のメンバーに感謝します。資金は、保健のNaitonal研究所の学内研究プログラムによって提供されていました。

1。マウス胚の四肢の皮膚の収集（E13.5〜E17.5） <ol><li>安楽死は、承認された手順によって女性を差し込む。私達の承認された動物のプロトコルによると、女性はCO 2曝露により安楽死され、その後、頚椎脱臼により保証。その吸水性のペーパータオルに動物を置くと、スクイーズボトルから70％エタノール/ H 2 Oでそれを徹底的に浸します。 </li><li>子宮はそのままに摘出し、血液を洗い流すために、氷冷ハンクス平衡塩溶液（HBSS）を含む100 × 15ミリメートルペトリ皿に入れてください。 </li><li>胚を分離して分析する。胚から非常に薄い羊膜を取り外します。 </li><li>各胚は遺伝子型をする必要がある場合（オプション）35 x 10 mmのシャーレに、単一の胚を摘出。解剖テールは、ジェノタイピングのために0.2ミリリットルのPCRチューブに移している。 </li><li>胚の前肢を切断し、転送はリン酸緩衝生理食塩水（PBS）で氷冷新鮮な4％パラホルムアルデヒド（PFA）の2 mlを入れた24ウェルプレートにリングの鉗子によって前肢。 </li><li> 4℃で一晩Nutatorミキサーで穏やかに混合して前肢を固定してください。 </li><li>翌日に、PFAを削除し、室温でNutatorミキサーで穏やかに混合したPBSを2 ml中で5分間3回洗浄する。 </li><li> 100％メタノール（MeOH）で前肢を移し、-20℃酵素の冷凍庫（臨界温度制御付きと自動霜取り機能のない冷凍庫）で保管してください。一次抗体は、100％メタノール処理後の表1の作業にリストされています。 </li><li>細かいピンセットを使用して前肢から剥がれ皮膚。手足の皮膚は、乾燥、手足から容易に分離する必要があります。最初に上に向けて腹側（手のひら側）で手足を置く。ビデオに示されるようにし細かいピンセットを使って、皮膚を​​カット。次のいずれかのダメージを与えることなく穏やかに肌を剥離、全体の肢の周囲解剖。 </li></ol> 2。リムのスキンのホールマウント免疫組織化学的染色<ol><li> 5分ごとのMeOH / PBT（PBS + 0.2％トリトンX - 100）（75％、50％、25％）の段階的な一連インキュベートすることにより、5ミリリットルポリプロピレン丸底チューブの手足の皮を水和。ソリューションを交換すると四肢の皮を損傷しないように気をつけなければならないことに注意してください。 </li><li>室温でNutatorミキサーで穏やかに混合してPBTで5分間2回洗浄する。 </li><li>でNutatorミキサーで穏やかに混合して2時間ヤギ二次抗体またはロバ二次抗体を10％ロバ血清/ PBS 0.2パーセントTX100ブロッキング緩衝液を10％ヤギ血清/ PBS 0.2パーセントTX100ブロッキング緩衝液のいずれかと四肢のスキンをブロック室温。 </li><li>ブロッキングバッファーで一次抗体の800μl（表に記載されているように、適切な希釈）（と2ミリリットル-マイクロ遠心チューブにリングの鉗子で35 x 10 mmのペトリ皿および転送に関する手足の皮を置き、いずれかの10％ヤギ血清/ PBS 0.2 ％TX100または10％ロバ血清/ PBS 0.2パーセントTX100）。 4℃で一晩Nutatorミキサーで穏やかに混合して四肢のスキンをインキュベートする。別の種（例えば、ラットモノクローナル抗体+ウサギポリクローナル抗体）から派生した複数の一次抗体を同時に使用することができることに注意してください。 </li><li>次の日に、2％ヤギ血清/ PBS 0.2パーセントTX100または2のどちらかは、洗浄バッファー（の4ミリリットルで5ミリリットルポリプロピレン丸底チューブにリングの鉗子で35 x 10 mmのペトリ皿や転送に四肢のスキンを配置％ロバ血清/ PBS 0.2パーセントTX100）。 </li><li>穏やかな室温でNutatorミキサーで混合しながら15分間5回洗浄する。 </li><li>ブロッキングバッファー中の二次抗体の800μl（どちらか10％ヤギ血清/ PBS 0.2パーセントTX100をまたは10％ロバ血清/を持つ2ミリリットル、マイクロ遠心チューブにリングの鉗子で35 x 10 mmのペトリ皿や転送に四肢のスキンを配置PBS 0.2パーセントTX100）。一般的に我々は、ジャクソンイムノ（Cy3標識、Cy5で、1:300希釈）またはInvitrogen社（のAlexa Fluor ® 488のAlexa Fluor 568、のAlexa Fluor 633、1:250希釈）から二次抗体を使用してください。二次抗体の凝集した粒子を除去するために0.22μmのPVDF膜シリンジフィルターを用いて二次抗体溶液をフィルタ。室温でNutatorミキサーで穏やかに混合して1時間暗いまたはアルミホイルで包んだの四肢のスキンをインキュベートする。異なる種に由来する異なる蛍光標識二次抗体を同時に使用することができることに注意してください。 </li><li>洗浄バッファーの4ミリリットル（2％ヤギ血清/ PBS 0.2パーセントのどちらかTX100または2％ロバ血清/ PBSで5ミリリットルポリプロピレン丸底チューブにリングの鉗子で35 x 10 mmのペトリ皿や転送に四肢のスキンを配置0.2パーセントTX100）。暗いまたは穏やかな室温でNutatorミキサーで混合とアルミ箔で包んで15分間5回洗浄する。 </li><li> （核に対する対比のためのオプション）インビトロジェンT3605 TO - PRO - 3（洗浄バッファー（2％ヤギ血清/ PBS 0.2パーセントTX100または2％ロバ血清/ PBSのいずれか0.2パーセントTX100）の4ミリリットルと四肢のスキンをインキュベート、暗いまたは部屋temperatuでNutatorミキサーで穏やかに混合しながら10分間アルミホイルで包んで1:3000希釈）再。その後、上ミキシング優しいと暗いまたはアルミホイルでラップされたの洗浄バッファー（2％ヤギ血清/ PBS 0.2パーセントTX100または2％ロバ血清/ PBSのいずれか0.2パーセントTX100）の4ミリリットルで5分間3回洗浄室温でNutatorミキサー。原子核の強いと特異的染色が特定の排出（HeNeレーザ633nmの励起）でTO - PRO - 3で観察されていることに注意してください。 </li></ol> 3。スライド上のリムのスキンの取り付け<ol><li> 35 x 10 mmのシャーレの上で手足の皮を置きます。粉塵、結晶、漂白豊富な写真を避けるために、低照度で実体顕微鏡下で微細なピンセットを使用してスキンの内側の層から繊維を取り除く。 </li><li>リングの鉗子によって接着剤顕微鏡スライドに手足の皮を転送します。スライド（カバーガラスに向かってすなわち）に上向きに横たわって内層でのスキンを置きます。慎重に細かいピンセットを使ってスキンを平らにし、キムワイプで洗浄バッファーキャリーオーバーを削除します。 </li><li>気泡なしでアンチフェードマウントメディアにマウントします。我々は、25&quot;X25&quot;カバースリップを使用してください。暗闇の中で平らな場所に置き、乾燥（例えば、サンプルが表示する前に、室温で暗所で一晩置かれているゴールド試薬を延長使用してマウント）。長期保存のために、4でスライドして格納するためにカバースリップをシール℃に</li></ol> 4。共焦点顕微鏡<ol><li>蛍光体の適切なレーザーを設定します。我々は、アルゴン488nmの（のAlexa Fluor ® 488およびGFPの場合）、DPSS 561nm（のAlexa Fluor 568およびCy3用）とヘリウムネオン633nm（のAlexa Fluor 633、Cy5標識およびTO - PRO - 3）を含む3つのレーザー源とライカTCS SP5共焦点顕微鏡を使用してください。 </li><li>ダブルまたはトリプル染色サンプルのすべての色素を同時に励起されるのクロストークを軽減または回避するシーケンシャルスキャンツールを使用してください。シーケンシャルスキャンモードでは、画像を順番に記録されます。 </li><li>励起用の蛍光色素とレーザーに関する一般的な情報はヒブズによる&quot;生物学者のための共焦点顕微鏡&quot;（2004）年に設立されることがあります</li></ol> 5。代表的な結果汎血管内皮細胞のマーカーに対する抗体とE15.5のマウス前肢の皮膚のホールマウントトリプルラベル共焦点immnofluorescence顕微鏡PECAM - 1（図1A - C、Dブルー）、ニューロフィラメントマーカー2H3（図1A緑）、および平滑筋細胞マーカーαSMA（図1A、B、赤）は、四肢の皮膚に2H3 +末梢神経に沿ったαSMA+動脈、の特徴的な分岐パターンを明らかにした。分岐血管に加えて、この四肢の皮膚の血管系のモデルは、リンパ管内皮細胞マーカーLYVE - 1（図1D、E赤）とNeuropilin2（NRP2）（図1D、F緑に対する抗体を用いて分岐リンパ管のパターニングを研究するために使用され）。 <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="図1" /> 図1（AC）動脈は、胚肢の皮膚の末梢神経に合わせます。汎血管内皮細胞マーカーPECAM - 1（AC、青）に対する抗体とホールマウントトリプルラベル共焦点immnofluorescence顕微鏡、ニューロフィラメントマーカー2H3（A、緑）、および平滑筋細胞マーカーαSMA（AとB、赤）が表示されます。 E15.5、2H3で+末梢神経（オープン矢印は）αSMAでカバーされている動脈（矢頭）+平滑筋細胞に関連付ける。胚肢の皮膚の（DF）リンパ脈管。汎血管内皮細胞のマーカーに対する抗体PECAM - 1（D、青）、リンパ管内皮細胞マーカーLYVE - 1（DとE、赤）とNeuropilin2（NRP2）（DとF、緑とトリプルラベル共焦点immnofluorescence顕微鏡）が表示されます。 LYVE - 1はまた、マクロファージ（オープン矢頭）の部分集合で表されるのに対し、リンパ管は、LYVE - 1とNRP2両方により可視化。スケールバー：100μmの。

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

利害の衝突は宣言されません。

それが臓器に十分な酸素と栄養を供給するので、血管系は、胚形成の間だけでなく、臓器のメンテナンスと成人の生殖機能のための器官の開発にとって極めて重要である。適切な血管網は、既存の毛細血管網が高度に分岐したと階層構造で編成されている血管新生により、複雑で多段階のプロセスで十分に確立されている。数々の作品は様々な分子がこ​​れらのプロセスに関与しているこ?...

抗体 汎血管内皮細胞のマーカー <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> PECAM - 1 </td><td>アルメニアハムスター（M） </td><td>ケミコン</td><td> MAB1398Z </td><td> 1:100希釈＃1 </td></tr><tr><td> PECAM - 1 </td><td>ラット（M） </td><td> BD </td><td> 553369 </td><td> 1:300希釈</td></tr><tr><td> VEGFR2 </td><td>ラット（M） </td><td>ベイバイオサイエンス</td><td> 14-5821-82 </td><td> 1:200希釈</td></tr><tr><td> CD34 </td><td>ラット（M） </td><td>ベイバイオサイエンス</td><td> 13から0341 </td><td> 1:300希釈</td></tr><tr><td>コラーゲンIV </td><td>ウサギ（P） </td><td> ABD Serotec </td><td> 2150-1470 </td><td> 1:300希釈＃2 </td></tr></tbody></table> 動脈内皮細胞のマーカー <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> Neuropilin1 </td><td>ウサギ（P） </td><td>アレックスKolodkinのラボ第3位 </td><td></td><td> 1:3000希釈</td></tr><tr><td> Unc5H2 </td><td>ヤギ（P） </td><td> R＆D </td><td> AF1006 </td><td> 1:200希釈</td></tr></tbody></table> 静脈内皮細胞のマーカー <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> EphB4 </td><td>ヤギ（P） </td><td> R＆D </td><td> AF446 </td><td> 1:100希釈</td></tr></tbody></table> リンパ管内皮細胞マーカー <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> LYVE - 1＃4 </td><td>ウサギ（P） </td><td>アブカム</td><td> ab14917 </td><td> 1:200希釈</td></tr><tr><td> LYVE - 1＃4 </td><td>ラット（M） </td><td> MBL </td><td> D225 - 3 </td><td> 1:200希釈</td></tr><tr><td> PROX - 1 </td><td>ウサギ（P） </td><td>ケミコン</td><td> AB5475 </td><td> 1:1000希釈</td></tr><tr><td> PROX - 1 </td><td>ヤギ（P） </td><td> R＆D </td><td> AF2727 </td><td> 1:50希釈</td></tr><tr><td> Neuropilin2 </td><td>ウサギ（P） </td><td>細胞シグナリング</td><td> 3366 </td><td> 1、100希釈</td></tr><tr><td>ポドプラニン</td><td>シリアンハムスター（M） </td><td>ハイブリドーマバンク</td><td> 8.1.1 </td><td> 1:200希釈</td></tr></tbody></table> 平滑筋細胞/周皮細胞マーカー <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> αSMA- CY3 </td><td>マウス（M）＃5 </td><td>シグマ</td><td> C - 6198 </td><td> 1:500希釈＃6 </td></tr><tr><td> NG2 </td><td>ウサギ（P） </td><td>ケミコン</td><td> AB5320 </td><td> 1:200希釈</td></tr><tr><td> SM22α </td><td>ウサギ（P） </td><td>アブカム</td><td> ab14106 </td><td> 1:200希釈</td></tr></tbody></table> GFPレポーターのための抗体 <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> GFP </td><td>ウサギ（P） </td><td>インビトロジェン</td><td> A11122 </td><td> 1:300希釈</td></tr><tr><td> GFP </td><td>ラット（M） </td><td>ナカライテスク</td><td> 04404〜84 </td><td> 1:1000希釈</td></tr><tr><td> GFP </td><td>チック（P） </td><td>ケミコン</td><td> P42212 </td><td> 1:300希釈</td></tr></tbody></table> LacZレポーターのための抗体 <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> β- galを</td><td>ウサギ（P） </td><td> MPバイオメディカル</td><td> 55976 </td><td> 1:5000希釈</td></tr><tr><td> β- galを</td><td>ヤギ（P） </td><td> ABD Serotec </td><td> 4600-1409 </td><td> 1:500希釈</td></tr><tr><td> β- galを</td><td>チック（P） </td><td>アブカム</td><td> ab9361 </td><td> 1:200希釈</td></tr></tbody></table> 末梢軸索のための抗体 <table border="1"><tbody><tr><td> 抗体 </td><td> 種 </td><td> 会社 </td><td> カタログ＃ </td><td> 労働条件 </td></tr><tr><td> 2H3 </td><td>マウス（M）＃7 </td><td>ハイブリドーマバンク</td><td> 2H3 </td><td> 1:200希釈</td></tr><tr><td> Tuj1 </td><td>マウス（M）＃8 </td><td> Covance </td><td> MMS - 435P </td><td> 1:500希釈</td></tr><tr><td>ペリフェリン</td><td>ウサギ（P） </td><td>ケミコン</td><td> AB1530 </td><td> 1:1000希釈</td></tr></tbody></table> シュワン細胞を移行するための抗体 <table border="1"><tbody><tr><td> BFABP </td><td>ウサギ（P） </td><td>トーマスミュラーの研究室第9位 </td><td></td><td> 1:3000希釈</td></tr></tbody></table> （P）：ポリクローナル抗体、（M）：モノクローナル抗体第1位：ヤギ抗アルメニアハムスター- Cy3の（ジャクソンイムノ127-165-160）抗体を二次抗体として使用する必要があります。 第2位：コラーゲンIV抗体は、in situハイブリダイゼーションの後に血管を検出するために使用することができます。 ＃3：Neuropilin1の抗体は、親切にジョンズホプキンス大学のアレックスKolodkinの研究室によって提供されます。我々はまだそれをテストしていないもののヒツジ抗ヒトNeuropilin1の抗体は、R＆D（AF3870）で利用可能です。 ＃4：LYVE - 1抗体はまた、胚性皮膚におけるマクロファージのサブセットを検出する。 ＃5：抗αSMA抗体マウスIgG2aモノクローナル抗体です。 ＃6：Cy3結合αSMAの抗体は、秒と一緒に室温で1時間インキュベートする他の一次抗体のための二次抗体。 ＃7：2H3抗体神経フィラメントに対するマウスIgG1モノクローナル抗体です。 ＃8：Tuj1抗体はニューロン固有のクラスIIIβ-チューブリンに対するマウスIgG2aモノクローナル抗体である。 ＃9：BFABP（脳特異的脂肪酸結合タンパク質）抗体は、親切に分子医学のマックスデルブリュック - センターのトーマスミュラーの研究室によって提供されます。

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

イメージングのためのホールマウント免疫組織化学分析は、全​​体の血管系は分岐形態形成の細胞メカニズムを理解するために極めて重要である。我々は、既存のプリミティブ毛細血管叢が階層的に分岐した血管網に再編成されている血管の発達を研究するために四肢の皮膚の血管系モデルを開発した。複数のラベルを持つホールマウント共焦点顕微鏡は、特定の蛍光マーカーを使用して、無傷の血管の堅牢な画像だけでなく、内皮細胞、周皮細胞および平滑筋細胞を含むそれらの細胞成分が可能になります。遺伝学的研究でこの四肢の皮膚の血管系モデルの進歩により、血管の開発とパターン形成の分子機構を理解改善している。四肢の皮膚の血管系のモデルは、末梢神経、動脈の分化とパターン形成のために空間的なテンプレートを提供する方法を研究するために使用されています。このビデオの記事では、我々は血管の開発のプロセスに従うことができる血管新生化胚の臓器に適用可能な血管特異的な抗体と蛍光二次抗体、で無傷の血管を染色するためのシンプルで堅牢なプロトコルを記述します。

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

我々はマウス胚の四肢の皮膚に複雑な血管網の形成を分析するために複数のラベルで共焦点顕微鏡を走査型ホールマウントの免疫組織化学とレーザーをご紹介。

胚肢の皮膚の血管系のためのホールマウント免疫組織化学的解析：胚における血管分岐形態形成を研究するモデルシステム

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K ビュー.  National Heart, Lung, and Blood Institute, National Institutes of Health. 我々はマウス胚の四肢の皮膚に複雑な血管網の形成を分析するために複数のラベルで共焦点顕微鏡を走査型ホールマウントの免疫組織化学とレーザーをご紹介。

ビデオ: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

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

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

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 ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

マウス胎児肺の文化、分岐の分子機構を評価するシステム

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

無血清全胚培養系で、マウス胚発生

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

活性化カスパーゼ3を検出するために、ホールマウント免疫蛍光法によりゼブラフィッシュ胚におけるアポトーシスの分析

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...