室管理ありがとう k. ギル、テクニカル サポート、j. ホーキンスとスタッフの国立機関の健康 (NIH) マウス ・ r ・ リードとの援助のための 50 の動物実験施設と行政支援のため f. Baldrey を構築します。その耳は解剖のプロトコルを皮膚、編集のヘルプ、および幹細胞研究室のメンバーのため N. やけどや技術的なヘルプと思慮深い議論の神経血管生物学を共有するための s. 茂木と m. Udey にも感謝。拓山崎は、科学振興 (JSPS) NIH 海徳の日本社会によって支えられました。この作品は、国立心臓、肺、血液研究所 (y. m. に HL005702-11) の学内研究プログラムによって支えられました。

このセクションのすべての実験は、国立心臓、肺および血の協会 (NHLBI) 動物のケアおよび使用委員会の承認の下で行った。
1. アダルト マウス耳皮膚コレクション
<ol><li>密閉室内の二酸化炭素 (CO2) 暴露による成体を安楽死させるし、頚部転位による安楽死を確認します。 注: 実験安楽死法の国立衛生研究所 (NIH) のガイドラインに従った。</li>
	<li>ベースから耳を解剖し、2 mL のハンクのバランスの取れた塩ソリューション (HBSS) を含むシャーレ 35 x 10 mm2に配置。簡単にハサミで毛をトリミングします。</li>
	<li>後部の皮膚と、介在する軟骨から慎重に離れて前方の皮膚の皮をむきます。 注: 軟骨は前方の皮膚に付着します。</li>
	<li>後部を転送やよく 24 に分けて前方皮膚板含む 1 mL 冷たい新鮮な 4% パラホルムアルデヒド (PFA) リン酸緩衝生理食塩水 (PBS) ウェルあたりで。4% の後部と前部の皮膚を平らにする PFA。</li>
	<li>1 h の 4 ° C で軟骨の後部と前部の皮膚を修正します。</li>
	<li>穏やかな室温でミキサーの混合 1 mL の PBS で 3 時間 5 分の後部と前部の皮膚を洗浄します。</li>
	<li>軟骨の後部と前部の皮膚をシャーレ 35 x 10 mm2の下部に転送します。折り畳まれ、脂肪や結合組織を持っているベースの領域を遮断します。良いカーブタイプ鉗子を使用して前方の皮膚から軟骨をはがします。</li>
	<li>高級ピンセットを使用して後部の皮膚の内側から毛、脂肪組織や結合組織を注意深く取り外します。PBS に濡れた肌を保ちます。</li>
</ol>2. ホール マウント免疫組織染色マウス耳介皮膚
注: NIH 研究所安全ガイドラインに従って次のセクションですべての実験を行った。
<ol><li>ブロックのバッファーを準備します。0.2% triton X-100 (テキサス州-100) ブロック バッファーのフィルター 10% 熱不活化ヤギ血清 (HIGS) PBS で希釈または 0.45 μ m のフィルター ユニットを使用してテキサス州 100 ブロック バッファー 0.2% で PBS で希釈した 10% ロバ血清 (DS)。</li>
	<li>後部を転送し、前方の皮膚の 24 ウェル プレート含む 1 mL の 10 %higs ブロック バッファーかウェルあたり 10 %ds ブロック バッファー。室温でのミキサーで穏やかな混合と 30 分のため皮膚を孵化させなさい。</li>
	<li>一次抗体 (材料表) ブロック バッファーで希釈することによって一次抗体溶液を調製 (いずれか 10% HIGS 10 %ds)。 注: 全体 mout 免疫組織化学的解析パン神経マーカー ニューロン固有クラス III β-チューブリン抗体による大人の耳の皮膚の (Tuj1、ウサギ ポリクローナル IgG またはマウス monocloncal igg2a 産、2 μ G/ml の最終濃度 1: 500 希釈)パン血管内皮細胞マーカー血小板内皮細胞接着分子 1 (PECAM-1、ハムスター モノクローナル IgG、1: 300 3.3 μ G/ml の最終濃度に希釈)、髄鞘マーカー ミエリン塩基性タンパク質 (MBP、ウサギ ポリクローナル IgG、1: 200 で希釈、最終濃度 5 μ G/ml) と炎症性の骨髄球系細胞マーカー CD11b (ラット モノクローナル IgG2b, 1:50 最終 20 μ G/ml の濃度で希釈)図 1と図 2に示した。皮膚は血管平滑筋細胞マーカー α 平滑筋アクチン (αSMA) 二次抗体 (2.6) と共に、Cy3 標識抗体とインキュベートだった。自分でテストした一次抗体は、材料表に記載されました。種から派生した複数の一次抗体を同時に混合することができます。</li>
	<li>後方と前方の皮膚を一次抗体溶液 150 μ L を含む新しい井戸に転送します。一晩に 4 ° C でミキサー穏やかな混合肌を孵化させなさい。</li>
	<li>次の日に 24 well プレートに新しい井戸を後部と前部の皮膚を転送または一次抗体を含むブロック バッファーを吸引します。PBS 0.2% テキサス州 100 洗浄バッファーまたは 2% 0.2% テキサス州 100 洗浄バッファーで PBS で希釈した DS で HIGS 希釈いずれか 2 %1 mL を追加します。洗浄液室温でミキサーで穏やかな混合と 15 分毎の 3 変化で皮膚を洗ってください。</li>
	<li>二次抗体の解決 (資材表) を準備します。ブロック バッファー内の二次抗体を希釈 (いずれか 10% HIGS 10 %ds) と 0.22 μ m ポリフッ化ビニリデン (PVDF) 二フッ化膜シリンジ フィルターを通して二次抗体を含むブロックのバッファーを 1 mL の注射器に接続されているフィルター。 注: Alexa 488 または 633 共役ヤギ抗うさぎ IgG (H + L) マウス IgG2a PECAM 1、アレクサ 488 標識ヤギ抗ウサギ IgG (H + L)、MBP のための Alexa 647 標識ヤギ抗ハムスター IgG (H + L) Tuj1 や Alexa 594 共役ラット IgG (H + L) CD11b のために使用されました。1: 250 最終 8 μ G/ml の濃度で希釈。皮膚は、Cy3 共役 αSMA 抗体 (最終濃度 2 〜 3 μ g/mL で希釈 1: 500) の組み合わせでこれらの二次抗体とインキュベートだった。</li>
	<li>ブロック バッファーから二次抗体の粒子を削除する 10 分 13,000 x g で溶液を遠心します。 注: 種から派生した異なる蛍光共役二次抗体を同時に混合することができます。</li>
	<li>まあ二次抗体溶液 150 μ L を含む後部と前部の皮膚に転送します。光を避けるために、1 時間室温でミキサーに穏やかな混合肌をインキュベートするアルミ箔で 24 well プレートをラップします。</li>
	<li>24 well プレートに新しい井戸を後部と前部の皮膚を転送したり完全にピペットで二次抗体溶液を吸引します。2 %higs 洗浄液または 2 %ds 洗浄液の 1 mL を追加します。</li>
	<li>24 well プレートをアルミ箔で包むし、洗う洗浄バッファーの 3 つの変更と室温でのミキサーで穏やかな混合と 15 分毎。</li>
</ol>3. スライドの耳介皮膚をマウント
<ol><li>皮膚をシャーレ 35 x 10 mm2の下部に配置します。広汎な写真の漂白を最小限に抑える低照明顕微鏡下の微細カーブタイプ鉗子を使用して肌の内側から毛、脂肪組織、結合組織、粉塵、繊維を慎重に取り外します。PBS に濡れた肌を保ちます。</li>
	<li>皮膚を鉗子を使用して剥離防止用スライドに転送します。内側の後部と前部の皮膚を置くスライド上を向いてします。鉗子を使用して皮膚を平らにします。</li>
	<li>退色を避けるために、蛍光信号を保持液耐フェード取付中肌をマウントします。空気の泡に上または皮膚の周りされていないことを確認します。</li>
	<li>皮膚サンプル coverslip 慎重にカバーし、メディアをマウントをしっかり得るように常温で暗い夜間にマウントされた皮膚サンプル スライドを保存します。マニキュアとスライドする coverslip を密封し、長期保存のための 4 ° C で保存します。</li>
</ol>4. 共焦点顕微鏡
<ol><li>Fluorophores の適切なレーザーを設定します。3 つのレーザー ソース (アルゴン 488 nm、DPSS 561 nm および HeNe 633 nm) と共焦点顕微鏡は、この実験で使用されます。</li>
	<li>同時に避けるために、すべての重複を減らすのトリプル染色サンプルを興奮させる逐次スキャン ツールを使用します。 注: 画像は逐次スキャン モードを使用して連続的に行われます。</li>
	<li>対物レンズ 10 倍の下のイメージ。タイルのスキャン ツールを使用して全体の耳介皮膚をキャプチャします。Z スタックを設定し、z 位置が耳の皮の全体の厚さをカバーするかどうかを確認します。</li>
</ol>

<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., James, J., Nam, J., Uchida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+confocal+microscopy+for+vascular+branching+morphogenesis.">Whole-mount confocal microscopy for vascular branching morphogenesis.</a> Methods Mol Biol. 843, 69-78 (2012).</li><li>Li, W., Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemical+analysis+for+embryonic+limb+skin+vasculature:+a+model+system+to+study+vascular+branching+morphogenesis+in+embryo.">Whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo.</a> J Vis Exp. , (2011).</li><li>Yamazaki, T., 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=Tissue+Myeloid+Progenitors+Differentiate+into+Pericytes+through+TGF-beta+Signaling+in+Developing+Skin+Vasculature.">Tissue Myeloid Progenitors Differentiate into Pericytes through TGF-beta Signaling in Developing Skin Vasculature.</a> Cell Rep. 18, 2991-3004 (2017).</li><li>Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel-dependent+recruitment+of+sympathetic+axons:+looking+for+innervation+in+all+the+right+places.">Vessel-dependent recruitment of sympathetic axons: looking for innervation in all the right places.</a> J Clin Invest. 124, 2855-2857 (2014).</li><li>Li, W., 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=Peripheral+nerve-derived+CXCL12+and+VEGF-A+regulate+the+patterning+of+arterial+vessel+branching+in+developing+limb+skin.">Peripheral nerve-derived CXCL12 and VEGF-A regulate the patterning of arterial vessel branching in developing limb skin.</a> Dev Cell. 24, 359-371 (2013).</li><li>Chang, H., Wang, Y., Wu, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flat+mount+imaging+of+mouse+skin+and+its+application+to+the+analysis+of+hair+follicle+patterning+and+sensory+axon+morphology.">Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology.</a> J Vis Exp. , e51749 (2014).</li><li>Salz, L., Driskell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+Whole+Mount:+A+Novel+Processing+and+Imaging+Protocol+for+Thick,+Three-dimensional+Tissue+Cross-sections+of+Skin.">Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin.</a> J Vis Exp. , (2017).</li><li>Liakath-Ali, K., 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=Novel+skin+phenotypes+revealed+by+a+genome-wide+mouse+reverse+genetic+screen.">Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen.</a> Nat Commun. 5, 3540 (2014).</li><li>Gunawan, 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=The+methyltransferase+Ezh2+controls+cell+adhesion+and+migration+through+direct+methylation+of+the+extranuclear+regulatory+protein+talin.">The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.</a> Nat Immunol. 16, 505-516 (2015).</li><li>Avci, P., 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=Animal+models+of+skin+disease+for+drug+discovery.">Animal models of skin disease for drug discovery.</a> Expert Opin Drug Dis. 8, 331-355 (2013).</li><li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">Animal models of atopic dermatitis.</a> J Invest Dermatol. 129, 31-40 (2009).</li><li>Wagner, E. F., Schonthaler, H. B., Guinea-Viniegra, J., Tschachler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoriasis:+what+we+have+learned+from+mouse+models.">Psoriasis: what we have learned from mouse models.</a> Nat Rev Rheumatol. 6, 704-714 (2010).</li><li>Nunan, R., Harding, K. G., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+challenges+of+chronic+wounds:+searching+for+an+optimal+animal+model+to+recapitulate+their+complexity.">Clinical challenges of chronic wounds: searching for an optimal animal model to recapitulate their complexity.</a> Dis Model Mech. 7, 1205-1213 (2014).</li><li>O'Brien, P. D., Sakowski, S. A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+diabetic+neuropathy.">Mouse models of diabetic neuropathy.</a> ILAR J. 54, 259-272 (2014).</li><li>Yamazaki, T., 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=Whole-Mount+Adult+Ear+Skin+Imaging+Reveals+Defective+Neuro-Vascular++Branching+Morphogenesis+in+Obese+and+Type+2+Diabetic+Mouse+Models.">Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.</a> Sci Rep. 8, (2018).</li></ol>

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このプロトコルでは、大人の耳の皮膚神経血管構造と免疫細胞分布の分析のための全体マウント immunonohistochemical イメージングについて説明します。このメソッドは分岐の形態形成と末梢神経や血管のパターンだけでなく、免疫細胞と髪を含む皮膚成分の三次元分布を研究する研究者のための多くの実験的利点と考えています卵胞。さらに定量分析のためのイメージング ソフトウェアを使用?...

皮膚は 3 つの層で構成されています: 表皮、真皮および皮下。それは、研究開発と同様に再生、腫瘍、および大人の炎症の形態形成、分化、幹細胞維持にモデル システムとして使用されています。皮膚はさまざまな血管や末梢神経系と血管系の開発がとれたように支配されています。
以前そのままの末梢神経や血管、細胞成分1,2,3,を含む研究に複数付け全体マウント胚皮膚イメージングを実現しました。4: 感覚軸索、交感神経の軸索、シュワン神経細胞、内皮細胞、周、血管での血管平滑筋細胞 (VSMCs)。血管新生、中にプライマリ毛細血管網は集中血管リモデリングを受け、階層型血管分岐ネットワークに発展します。発展途上の真皮・皮下、動脈末梢感覚神経と静脈と一緒に分岐し、動脈に隣接して形成します。階層型の血管のネットワークは、VSMCs で覆われて徹底的に、交感神経に沿って延びるし、大径血管1,5,6を支配します。密接に関連開発神経系と血管系の意義、にもかかわらず主要な質問は大人でさまざまな病的状況で神経・血管のネットワークはどう対処してきました。三次元高分解能イメージングは、解剖学的に認識可能な形態とパターニングの病因を理解する必要です。
アダルト マウス皮膚の神経と血管の形態形成をよくティッシュ セクションの汚損によって解析しました。他の研究は、末梢神経、毛包、皮脂腺、arrector pili 筋肉7,8,9に加えて、血管を可視化するのに皮膚の全体マウント画像を使用しています。ただし、成人の皮膚の厚さは、その全体の深さで皮膚を分析することは困難が。
本研究では、大人の耳皮膚これらの課題を克服するための新高解像度全取り付けるイメージングを開発しました。耳の皮膚はその全体の深さを郭清と皮膚の後続の全体マウント イメージングの容易にアクセス可能です。したがって、包括的な定量化測定で、皮膚の末梢神経系と血管系の三次元のアーキテクチャを比較に適用することができます簡単で再現性の高い方法です。大人の肌に大径血管と末梢の感覚と交感神経神経の配置が保持されることを行った。このプロトコルの目標分岐の形態形成と末梢神経、血管、炎症など様々 な条件下で成体マウス モデルにおける細胞レベルでの免疫細胞の分布のパターンを視覚化することですし、再生。

<table><tbody><tr><td>10 x Phosphate Buffered Saline</td><td>KD Medical</td><td>RGE-3210</td><td>PBS, without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Gibco</td><td>14025-092</td><td>HBSS, with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA, fixative, diluted in PBS</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td>Detergent</td></tr><tr><td>Normal goat serum</td><td>Gibco</td><td>16210064</td><td>Component of blocking/washing buffer</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immuno research</td><td>017-000-121</td><td>Component of blocking/washing buffer</td></tr><tr><td>Curved fine tweezers</td><td>Dumont</td><td>RS-5047</td><td></td></tr><tr><td>Curved tweezers</td><td>Integra Miltex Vantage</td><td>V918-782, V918-784</td><td></td></tr><tr><td>Filter Unit 0.45 mm</td><td>Thermo Scientific</td><td>157-0045</td><td>For filtration</td></tr><tr><td>1 mL syringe</td><td>Coviden</td><td>8881501400</td><td>For filtration</td></tr><tr><td>Syringe filter Unit 0.22 mm</td><td>Millex-GV</td><td>SLGVR04NL</td><td>For filtration</td></tr><tr><td>ProLong Gold</td><td>Thermo Scientific</td><td>P36934</td><td>Anti-fade mounting medium</td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td>For sealing</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Photoshop CC 2017</td><td>Adobe</td><td></td><td>Graphics editor software</td></tr><tr><td>Illustrator CC 2017</td><td>Adobe</td><td></td><td>Graphics editor software</td></tr><tr><td>Image J</td><td>NIH</td><td></td><td>Image processing software</td></tr><tr><td>Anti-PECAM-1 (CD31) antibody</td><td>Millipore</td><td>MAB1398Z</td><td>Hamster IgG, vascular endothelial cell marker, 1:300</td></tr><tr><td>Anti-PECAM-1 (CD31) antibody</td><td>BD Pharmingen</td><td>553369</td><td>Rat IgG2a kappa, vascular endothelial cell marker, 1:300</td></tr><tr><td>Anti-&amp;#945;SMA antibody conjugated with cy-3</td><td>Sigma</td><td>C6198</td><td>Mouse IgG2a, vascular smooth muscle cell marker, 1:500</td></tr><tr><td>Anti-EphB1 antibody</td><td>Santa Cruz</td><td>sc-9319</td><td>Goat polyclonal, venous endothelial cell marker, 1:100</td></tr><tr><td>Anti-neuron-specific Class III &amp;#946;-tubulin (Tuj1)</td><td>Abcam</td><td>AB18207</td><td>Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500</td></tr><tr><td>Anti-Tuj1 antibody</td><td>Covance</td><td>MMS-435P</td><td>Mouse IgG2a, pan-axonal marker, 1:500</td></tr><tr><td>Anti-MBP antibody</td><td>Abcam</td><td>AB40390</td><td>Rabbit polyclonal IgG, myelination marker, 1:200</td></tr><tr><td>Anti-Tyrosine Hydroxylase antibody</td><td>Chemicon</td><td>AB152</td><td>Rabbit polyclonal, sympathetic neuron marker, 1:500</td></tr><tr><td>Anti-Peripherin antibody</td><td>Chemicon</td><td>AB1530</td><td>Rabbit polyclonal, peripheral neuron marker, 1:1000</td></tr><tr><td>Anti-CD11b antibody</td><td>Bio-Rad</td><td>MCA74G</td><td>Rat IgG2b, inflammatory cell marker (macrophages), 1:50</td></tr><tr><td>Anti-CD45 antibody</td><td>Thermo Fisher Scientific</td><td>14-0451-85</td><td>Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500</td></tr><tr><td>Anti-CD3 antibody</td><td>Bio-Rad</td><td>MCA1477T</td><td>Rat IgG1, immune cell marker, 1:100</td></tr><tr><td>Anti-CD45R (B220) antibody</td><td>Thermo Fisher Scientific</td><td>14-0452</td><td>Rat IgG2a kappa, inflammatory cell marker, 1:200</td></tr><tr><td>Anti-GFP antibody</td><td>Thermo Fisher Scientific</td><td>A11122</td><td>Rabbit polyclonal, 1:300</td></tr><tr><td>Anti-GFP antibody</td><td>Abcam</td><td>Ab13970</td><td>Chicken polyclonal, 1:500</td></tr><tr><td>Anti-&amp;#946;-gal antibody</td><td>Cappel</td><td>55976</td><td>Rabbit polyclonal, 1:5000</td></tr><tr><td>Anti-RFP antibody</td><td>Abcam</td><td>Ab62341</td><td>Rabbit polyclonal, 1:300</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa 488</td><td>Thermo Fisher Scientific</td><td>A11034</td><td>Rabbit polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-hamster IgG (H+L) Alexa 647</td><td>Jackson Immuno research</td><td>127-605-160</td><td>Hamster polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-rat IgG (H+L) Alexa 594</td><td>Jackson Immuno research</td><td>112-585-167</td><td>Rat polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-mouse IgG2a Alexa 633</td><td>Thermo Fisher Scientific</td><td>A21136</td><td>Mouse IgG2a secondary antibody, 1:250</td></tr></tbody></table>

whole-mount confocal microscopy for adult ear skin: a model system to study neuro-vascular branching morphogenesis and immune cell distribution

ここでは、包括的な三次元神経血管の分岐形態形成、パターニング、細胞レベルでの免疫細胞の分布を研究するイメージング全体マウント大人の耳皮のプロトコルを提案します。成体組織の末梢神経や血管の解剖学的構造の解析は、神経血管機能の配線と創傷治癒など病態における神経血管変性の理解にいくつかの洞察を提供します。非常に有益なモデル システムとして解離しやすく、大人の耳の皮膚に私たちの研究をしました。私たちのシンプルで再現性のあるプロトコルが全身の皮膚、末梢神経 (感覚軸索、交感神経の軸索とシュワン細胞) など血管 (血管内皮細胞と血管平滑筋細胞の細胞内成分の正確な描写を提供します)、および炎症性細胞。このプロトコルは異なる病態下大人の耳の皮膚の炎症だけでなく、末梢神経や血管の形態異常を調査する道を開くだろうと考えています。

成体マウス後部耳介皮膚 (図 1 a)、前耳介皮膚 (図 1 b) が αSMA (赤), Tuj1 (緑)、抗体 immunostained と PECAM-1 (青)。後部の皮膚だった CD11b (赤) と MBP (緑)、Tuj1 (青) と (図 2 a) に対する抗体を用いた神経免疫分布を研究する immunostained です。CD11b の分布+単一セルの解像度 (図 2 b) で...

Watch this Scientific Journal Video about Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution at JoVE.com

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

ここでは、分岐の形態形成および末梢神経、血管、免疫細胞分布のパターンを視覚化することを可能にする全体の大人マウス耳介皮膚に高解像度全体マウント画像処理手法について述べる。

大人の耳の皮膚の共焦点顕微鏡を全取り付ける: 神経血管分岐の形態形成と免疫細胞の分布を検討するモデル システム

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

whole-mount-confocal-microscopy-for-adult-ear-skin-model-system-to

Research

JoVE Journal

Developmental Biology

13.1K ビュー.  National Institutes of Health. ここでは、分岐の形態形成および末梢神経、血管、免疫細胞分布のパターンを視覚化することを可能にする全体の大人マウス耳介皮膚に高解像度全体マウント画像処理手法について述べる。

ビデオ: Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

マウス皮膚や毛包パターニングおよび感覚軸索形態の解析への応用のフラットマウントイメージング

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

神経科学

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

炎症真皮における撮像CD4 T細胞間質への移行

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

免疫・感染学

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 ...

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

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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 ...

生物学

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

の毛包披針形語尾に神経末端標識リビングの光監視<em&gt; ex vivoで</em&gt;マウスの耳皮膚

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

マウス内耳組織の凍結切片および免疫染色:胚期から成虫期まで

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

発生生物学

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

免疫応答の生体内多光子イメージングのためのマウスの耳の皮膚に虚血再灌流傷害を誘導します

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

医学

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

マウスの洞房結節と房室結節のホールマウント免疫蛍光染色、共焦点イメージング、3D再構成

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Stria Vascularisの微小解剖による単一核シーケンシングまたは免疫染色

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

著者スポットライト:単一核シーケンシングまたは免疫染色のための成体マウス血管筋の解剖  

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

大人の耳の皮膚の共焦点顕微鏡を全取り付ける: 神経血管分岐の形態形成と免疫細胞の分布を検討するモデルシステム

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大人の耳の皮膚の共焦点顕微鏡を全取り付ける: 神経血管分岐の形態形成と免疫細胞の分布を検討するモデル システム

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大人の耳の皮膚の共焦点顕微鏡を全取り付ける: 神経血管分岐の形態形成と免疫細胞の分布を検討するモデルシステム