この作品は、ウェルカムトラストと英国心臓財団によって資金を供給された。

特に断らない限り、すべてのステップは、室温で行われる。 1。産後目の摘出および固定<ol><li>その側に子犬安楽死マウスを置き、ハサミで目を覆っている皮膚を除去。 </li><li>目を摘出するためにはさみやピンセットを使用してください。 </li><li>摘出眼球下場所はさみ、視神経と周囲の組織をカットし、目を持ち上げて外します。室温で2倍PBSで構成された4％パラホルムアルデヒド（PFA）を含む24ウェルプレートに移す。 </li><li>それぞれの目は、10のために修正するために許可する - 10分 - 15分、その後5氷上で冷たい倍PBSに転送。固定度を確保するためにenucleationsとその後固定をずらすことが最善であるそれぞれの目のために等しくなります。 </li></ol> 2。産後マウス網膜の解剖<ol><li> disse下穴および場所を広げるためにカットされているプラ​​スチック製パスツールピペットを用いて2倍のPBSを含むペトリ皿に目を移す顕微鏡cting。 </li><li>鉗子やハサミを使って目の周りの任意の脂肪を取り除きます。 </li><li>ピアース鋭いハサミで角膜の端と角膜と虹彩の周りカットして捨てる。 </li><li>網膜と強膜の間に鉗子を挿入して、網膜を引き裂くないように注意して強膜を除去する。脈絡膜層も除去することができるが、この工程の間に生じる網膜の損傷がある場合は、脈絡膜層は、それが著しく免疫染色の品質に影響を与えるべきではないように残すことができる。 </li><li>レンズとピンセットを用いて硝子体液を削除します。 </li><li>その後すぐに網膜の中心（あるいは細かいハサミで硝子体血管の基部に中略）から引き離し細かい鉗子でアイの中心でそれらを一緒に集めることによってhyaloids血管を削除します。 </li><li>きれいなペトリ皿にPBSでカップ形の網膜をすすぐ。 </li><li>春scissoを用いた網膜の半径の約2/3に達して4から5放射状の切開を行いますRS &#39;花びら&#39;形状を作成する。 </li><li>網膜を平らにするためにパスツールピペットを用いてPBSをオフに描画した後、吸収紙の小片で余分PBSを除去します。 </li><li>追加のメタノールで完全に覆われており、その後、洪水まで、網膜の表面にドロップでゆっくりピペット寒い（-20°C）メタノールドロップ。これは、網膜を固定するのに役立ちますし、透過性を促進する。網膜は、白になります。 </li><li>穴を広げるためにカットされたプラスチック製パスツールピペットを用いて2 mlのチューブに移します。 </li><li>免疫染色を進める前に、少なくとも20分間冷メタノールで網膜のままにしておきます。ここで使用される抗体のうち、VE-カドヘリンは、メタノール固定網膜上で正常に使用することはできません。この場合、網膜は、ステップ9の後に免疫染色にまっすぐ進んでする必要があります。 </li><li>必要に応じて前まで染色まで数ヶ月の​​ために、網膜を-20℃でメタノールに格納することができます。 </li></ol> 3。網膜Vascuの免疫/イソレクチン染色lature <ol><li>慎重に削除/メタノールを留注ぎ、優しく（この段階で網膜は、上記の手順11から同じ2 mlのチューブに残っている）をPBSで簡単に網膜をすすいでください。 </li><li>パーマ/ブロック溶液100μl（PBS + 0.3％トリトン+ 0.2％BSA）を含むPBS、カバー網膜に適切な血清+ 5％（ 表1を参照）を取り外し、1時間軽く振る。 </li><li>パーマ/ブロックを取り外し、優しい4℃で一晩振とうしながら網膜をインキュベート100℃選択し、一次抗体の液（ 表1を参照）、および/ ​​またはIsolectinB4とC、パーマ/ブロックで希釈することにより調製すべて。 </li><li>抗体/イソレクチンを取り外し、PBS + 0.3％トリトン（PBSTX）における網膜4×10分を洗う。 </li><li> IsolectinB4-Alexa488又は直接標識1次抗体で染色した後に例えばいくつかのケースにおいては、二次抗体は必須ではなく、網膜する（ステップ7）を直接取り付けることができる。コンジュゲートしていない一次抗体は、適当なフッ素を100μl次に、ステップ3で使用されているとき自己消費二次抗体（PBSTXで1/200希釈）を加え、4℃または室温で4時間撹拌一晩インキュベート置する。 </li><li>二次抗体を除去し、2ミリリットルPBSTXにおける網膜4×15分を洗う。 </li><li>広い口径プラスチックパスツールピペットを用いてスライドに網膜を転送し、吸収剤、ティッシュで余分PBSTXを削除します。 50μlのを追加してマウント網膜は、網膜上にカバースリップと優しく位置に封入を延長。 </li><li>転送は、彼らが延長封入が設定できるように、フラットで光から保護されていることを確認し、4℃で一晩冷蔵庫にスライドします。 </li><li>共焦点顕微鏡または落射蛍光顕微鏡を用いた画像網膜。 </li></ol>

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473 (7347), 298-307 (2011).</li><li>Staton, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+for+assaying+angiogenesis+in+vitro+and+in+vivo.">Current methods for assaying angiogenesis in vitro and in vivo.</a> Int J. Exp. Pathol. 85 (5), 233-248 (2004).</li><li>Goodwin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+of+angiogenesis+for+assessment+of+angiogenic+and+anti-angiogenic+agents.">In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.</a> Microvasc. Res. 74 (2-3), 172-183 (2007).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+eye+enucleation+for+remote+high-throughput+phenotyping.">Mouse eye enucleation for remote high-throughput phenotyping.</a> J. Vis. Exp. (57), e3184 (2011).</li><li>Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+the+neonatal+vasculature+and+analysis+of+retinal+angiogenesis+in+mice.">Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.</a> Nat. Protoc. 5 (9), 1518-1534 (2010).</li><li>Allinson, K. R., Lee, H. S., Fruttiger, M., McCarty, J., Arthur, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+expression+of+TGFbeta+type+II+receptor+is+required+to+maintain+vascular+integrity+during+postnatal+development+of+the+central+nervous+system.">Endothelial expression of TGFbeta type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.</a> PLoS One. 7 (9), e39336 (2012).</li><li>Mahmoud, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+arteriovenous+malformations+in+the+absence+of+endoglin.">Pathogenesis of arteriovenous malformations in the absence of endoglin.</a> Circ. 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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

ここで紹介する方法は、血管新生の容易に定量化し、生理学的に適切なモデル作り、新生児マウスの網膜のステンドホールマウント標本の画像を取得するための簡単​​かつ効率的な方法を提供しています。最初は、マウスの目は、その小さなサイズと地球の形状により解剖するのは非常に困難になる可能性がありますので、いくつかの練習と良い解剖ツールは信頼性のある結果を得るため?...

血管新生は、既存の血管ネットワークから新たな血管の形成によって定義され、いくつかのシグナル伝達経路により調節される複雑な発達過程である。これらの最も顕著なのはVEGF経路である。 VEGFは、隣接血管の発芽血管新生の開始に虚血細胞及びリードによって解除される。芽新しい血管からの主要な内皮細胞は、VEGFの源に向かって手を差し伸べることを糸状仮足を作り出す &#39;先端&#39;セル、であり、 &#39;茎&#39;内皮細胞増殖が続いている。このように、活性化内皮細胞は移動し、それらが新しい血管の管を形成する無血管領域に向かって増殖する。血流をサポートするために、これらのチューブを安定化させるために、内皮細胞は、周皮細胞と新しい血管1を囲む平滑筋細胞と相互作用する。血管新生は胚と成長組織の血管新生のために不可欠です。しかし、それはまた、多くのpathologiに寄与する癌などのCAL障害;血管新生における欠陥は虚血性疾患に関連付けられているのに対し、。疾患を治療するための手段として血管形成を調節するための効果的な薬物治療の開発は非常に興味深いことである。例えば、効果的な抗血管新生因子は、血管新生促進戦略は虚血性疾患の治療に使用することができる一方で、癌の増殖を減少させる。したがって、血管新生のモデルは、より良い新たな血管の形成と血管の成長を高めるか、または減少させるための新たな治療戦略を開発するための規則を理解することが不可欠です。 血管新生研究の基本的な要素は、適切な血管モデルの選択です。 in vitroモデルの多くは、このような内皮細胞の遊走、増殖および生存2,3のような血管形成の基本的な手順を再現するように設計されている。トンが頻繁にin vitroアッセイで使用される、マトリゲルマトリックス上内皮細胞の分枝ネットワークの形成である彼は、内皮細胞に特異的なものではなく、また、通常は周細胞又は平滑筋細胞の支持体を組み込んでいない。このような胚様体アッセイ、大動脈リングアッセイおよび胎児の中足骨アッセイとして、マウスの器官培養を用いたex vivoで発芽血管新生の評価は追加の支持細胞の文脈における内皮細胞の血管新生の分析を行うことができます。しかし、これらのアッセイの主な制限は、分析のために限られたウィンドウを与え、血流の欠如、時間の経過とともに血管の退縮が挙げられる。したがって、in vivoモデルにおいて 、より正確に血管新生の調節を理解するために、例えば皮下に移植スポンジ又はマトリゲルプラグ、並びに後肢虚血モデルを用いて、マウスにおいて開発されたものとして必要とされている。しかし、これらのモデルは、新生血管の複雑な3Dアーキテクチャにより分析することが挑戦しています。これとは対照的に、ゼブラフィッシュ胚全体で血管新生を可視化するための優れたモデルを証明している生物4。しかし、マウスはマウス、多くの場合において好ましいモデル作り、進化的にはるかに密接にゼブラフィッシュより人間に関連しています。結果的に出生後のマウス網膜の血管新生、血管新生研究のための選択のよく特徴付けメソッドを表します。それは生理的な設定だけでなく、容易に適切な蛍光汚れを用いて可視化することができ、2D血管叢を提供するだけでなく。血管ネットワークのアーキテクチャ、内皮増殖、発芽、血管周囲の細胞動員と血管リモデリングは、すべて、この手法を用いて検討することができます。 新生仔マウスの網膜において、網膜血管の発達は出生後の最初の週に起こる。マウスは、人間とは違って、生まれつき目が見えないされ、網膜のみ生後血管なる。網膜血管に近い生後0日（P0）で視神経に網膜の中心部から発生し、高度に組織化を形成するために、血管新生によって開発約P8 5によって網膜周辺部に到達したdの血管叢。血管が徐々に介入して毛細血管網で動脈と静脈の定期的な交流のパターンを生成するために周辺に向かって視神経乳頭から外側に成長毛細血管叢との特性の方法で開発しています。網膜血管系は、このように、それが比較的容易にフラットマウント標本5における網膜血管構造を検討すること、彼らは基本的に2D平面であるものに育つように血管が簡単に識別できるような血管新生を研究するための理想的なモデルを提供します。数時間のex vivoで上内皮先端細胞応答の分析のための新生児マウスの網膜を単離するための方法は、06報告されている。代替的に、全体の網膜血管系および関連血管マーカーのより詳細な調査は、開発の異なる段階で固定網膜標本の免疫染色が必要である。 ここでは、提供する我々は、顕微鏡下で染色プロトコルを介した出生後の眼の初期摘出（また、7参照）から最終的な画像に、マウスの網膜血管叢の全体マウント染色に使用する信頼性と技術的にまっすぐ進む方法の詳細な説明。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Plastic pipettes 3 ml</td>
			<td>Scientific Laboratory Supplies</td>
			<td>PIP4210</td>
			<td>Remove tip with scissors to create a wide bore</td>
		</tr>
		<tr>
			<td>BD Falcon 24-well plates</td>
			<td>Scientific Laboratory Supplies</td>
			<td>353226</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Select Petri dishes TV 90 mm</td>
			<td>Scientific Laboratory Supplies</td>
			<td>SLS2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Histobond slides</td>
			<td>VWR</td>
			<td>631-0624</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips 22 x 22 mm</td>
			<td>VWR</td>
			<td>631-1336</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5</td>
			<td>World precision instruments</td>
			<td>14095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spring scissors 10.5 cm long, with straight 8 mm blades</td>
			<td>World precision instruments</td>
			<td>501235</td>
			<td>Used for enucleation</td>
		</tr>
		<tr>
			<td>Vannas scissors 8.5 cm long, with straight 7 mm blades, and 0.025 x 0.015 mm superfine tips</td>
			<td>World precision instruments</td>
			<td>500086</td>
			<td>Used for dissecting retina</td>
		</tr>
		<tr>
			<td>Superfine vannas scissors 8 cm long with straight 3 mm blades, and 0.015 x 0.015 mm superfine tips</td>
			<td>World precision instruments</td>
			<td>501778</td>
			<td>Used for dissecting retina</td>
		</tr>
		<tr>
			<td>crystal clear' tubes 2 ml</td>
			<td>Starlab</td>
			<td>E1420-2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Make up in 2x PBS and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Mounting reagent</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-alexa 594</td>
			<td>Invitrogen</td>
			<td>I21413</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-alexa 488</td>
			<td>Invitrogen</td>
			<td>I21411</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primary Antibodies</td>
		</tr>
		<tr>
			<td>Anti-NG2 (rabbit polyclonal )</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td>Detects pericytes</td>
		</tr>
		<tr>
			<td>Anti-GFAP (clone GA5, mouse monoclonal)</td>
			<td>Millipore</td>
			<td>MAB360</td>
			<td>Detects astrocytes</td>
		</tr>
		<tr>
			<td>Anti-Desmin (clone Y66, rabbit monoclonal)</td>
			<td>Millipore</td>
			<td>04-585</td>
			<td>Detects pericytes</td>
		</tr>
		<tr>
			<td>Anti-Collagen IV (rabbit polyclonal )</td>
			<td>Chemicon</td>
			<td>AB756P</td>
			<td>Detects vascular basement membrane</td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle actin-Cy3 (clone 1A4, mouse monoclonal)</td>
			<td>Sigma</td>
			<td>C6198</td>
			<td>Detects vascular smooth muscle cells.</td>
		</tr>
		<tr>
			<td>Anti-Endoglin (clone MJ7/18, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>550546</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>Anti-CD31 (clone MEC13.3, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>Anti-VE-Cadherin (clone 11D4.1, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>550548</td>
			<td>Detects endothelial cell junctions</td>
		</tr>
		<tr>
			<td>Anti-Alk1 (goat polyclonal)</td>
			<td>R&amp;D Systems</td>
			<td>AF770</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Secondary Antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11012</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-rat-Alexa488</td>
			<td>Invitrogen</td>
			<td>A11006</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-rat-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11007</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-mouse-Alexa488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-goat-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11058</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Microscopes</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Zeiss</td>
			<td>Stemi SV6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Zeiss</td>
			<td>Axiocam HRc</td>
			<td>Camera for dissection microscope</td>
		</tr>
		<tr>
			<td>Epifluorescent Microscope</td>
			<td>Zeiss</td>
			<td>Axioimager with Apotome</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Zeiss</td>
			<td>Axiocam HRm</td>
			<td>Camera for Axioimager</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

whole mount immunofluorescent staining of the neonatal mouse retina to investigate angiogenesis in vivo

血管新生は、既存の血管ネットワークから新規血管の発芽によって定義された新しい血管形成の複雑なプロセスである。血管新生は、臓器や組織の正常な発達ではなく、例えば、癌、失明や虚血性疾患などの血管形成を調節不全である、多くの疾患、だけでなく、重要な役割を果たしている。大人の生活の中で、血管が新生が病気に特異的に新しい血管の形成を試み、規制する新たな薬剤開発のための重要なターゲットであるので、一般的に静止しています。優れた血管新生を理解し、それを規制するための適切な戦略を開発するために、モデルが正確に関与する異なる生物学的工程を反映していることが必要である。動脈、静脈と毛細血管が出産後の最初の週の間に血管叢を形成するために開発するため、マウス新生児の網膜は、血管新生の優れたモデルを提供します。このモデルは、2つの - ジを有するという利点を有するmensional（2D）構造は、他の血管のネットワークの複雑な3D解剖と比較して簡単な解析を行う。出生後の異なる時点で網膜血管叢を解析することにより、顕微鏡下で血管新生の様々な段階を観察することが可能である。この記事ではイソレクチンと血管特異的な抗体で蛍光染色を用いたマウス網膜の血管構造を解析するための簡単​​な手順を示しています。

解剖後網膜は、フラットの花（ 図1）のようになります。上記で概説プロトコルを使用することにより、血管内皮細胞を抗α平滑筋アクチン（ 図2）を用いて可視化されるイソレクチンB4-Alexa488染色を用いて、血管筋細胞を支持することが分かる。 図2の5倍対物レンズを用いて低消費電力の表示は、網膜の血管の組織の概要を可能にする。また、上記のプ?...

Watch this Scientific Journal Video about Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo at JoVE.com

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

新生児マウスの網膜は血管新生を調節する別の遺伝子や薬剤の役割の調査を可能にし、血管新生のよく特徴付け生理学的モデルを提供<em生体内で&gt;</em&gt;コンテキスト。正確に血管叢を可視化する免疫蛍光染色は、研究のこれらのタイプの成功に極めて重要です。

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色<em生体内で&gt;</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

whole-mount-immunofluorescent-staining-neonatal-mouse-retina-to

Research

JoVE Journal

Neuroscience

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

45.3K ビュー.  Newcastle University. 新生児マウスの網膜は血管新生を調節する別の遺伝子や薬剤の役割の調査を可能にし、血管新生のよく特徴付け生理学的モデルを提供コンテキスト。正確に血管叢を可視化する免疫蛍光染色は、研究のこれらのタイプの成功に極めて重要です。

ビデオ: Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

ホールマウントの胎児神経系の可視化ショウジョウバエ胚

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

神経科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

における嗅覚受容ニューロンのホールマウント免疫標識<em&gt;ショウジョウバエ</em&gt;アンテナ

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

マウスの胚感覚軸索投影のホールマウントイメージング

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

成体マウス蝸牛のホールマウント解剖および免疫蛍光

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Deafferentatedマウス網膜のフラットマウントの準備中のスターバーストアマクリン細胞のパッチクランプ記録

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

大人のホールマウントの準備のためのシンプルなワンステップ解剖プロトコル<em&gt;ショウジョウバエ</em&gt;ブレインズ

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

経瞳孔二光子マウス網膜のin vivoイメージング

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

菌体、円周体、口蓋味覚芽の全山染色、可視化、分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...