著者には謝辞はありません。

すべての実験手順は、東京都大学機関動物管理委員会（IACUC）によって承認された。動物は、実験動物のケアおよび使用のためのガイドおよびカナダ動物飼育評議会の実験動物のケアおよび使用のためのガイドに従って収容および処理された。東京都大学フロンティアヘルスサイエンス学科Functional Morphology Laboratoryで飼育維持されている動物を使用しました。 1.動物のケアと住居<ol><li>閉鎖した繁殖コロニーからS. murinus 7頭（体重が70〜90gの雌3頭、雄4頭）を得る。 </li><li>紙ストリップを含む木製の巣箱を備えたプラスチックケージに離乳後（出生後20日）個別にS. murinusをケージに入れる。慣習的に調整された動物の部屋に入れてください：23-27℃、湿度コントロールなし、そして12:12時間の明暗サイクル。供給業者イーグル・マス・ペレットと水を自由に与えます。 </li></ol> 2.組織の準備<ol><li> 4％（w / v）パラホルムアルデヒド（PFA）を調製するために、PFA40gを0.01Mリン酸緩衝生理食塩水（PBS、pH7.4）1000mLに溶解する。ヒュームの棚で、溶液が透明になるまでボルテックスで混合する。 4％PFA O / Nは4℃で保管してください。 注意：PFAを取り扱う際は、適切な個人用保護具（PPE）を着用してください。 </li><li>動物を灌流して固定するために、それをエーテルで麻酔し、次にそれにsomnopentyl（ペントバルビタールナトリウム、0.6mL / kg体重）の腹腔内注射を与える。 </li><li>動物が完全に麻酔された後、メスで腹腔を開き、腹部の真ん中に3cmの長さの腹部切開部を作ります。腹部大動脈の切開部のすぐ上のレベルで腹部大動脈にカテーテルを腸骨動脈に挿入する。 </li><li>ソノノペンチルを注射する（ペントバルビタール体重1kgあたり1.0mL）。完全な安楽死後、0.01M PBS（pH 7.4）で灌流し、次いで煙霧棚で4％PFAで灌流する。 </li><li>灌流後、白いネオプレンラテックス（希薄白色ネオプレンラテックス3：2、蒸留水（DW））を2〜3mL注入して血管に標識する。 </li><li>肝臓、胆嚢、総胆管、十二指腸、膵臓などの腹部の器官を関連する神経や血管から抽出する。 </li><li>約12 mLの青色ネオプレンラテックス（希釈した白色ネオプレンラテックス10 mLに1滴の青色インクを加える）を胆嚢に注入して、胆管系を標識する。 </li><li>全マウント免疫染色のために、4℃で4％PFAで一晩固定してください。 注意：PFAは有毒です。 PFAの直接取り扱いは避けてください。 PFAはヒューム棚に使用する必要があります。 </li></ol> 3.全身免疫組織化学注：プロトコールを通して、洗浄、抗体インキュベーション、および着色を含む、the標本は、室温または4℃でゆっくりとロッキングして、ナットレーター上に留まらなければなりません。 <ol><li> 1日目：潜在的な内因性ペルオキシダーゼの酵素処理および不活性化 <ol><li>調製した試験片をDW 4xで室温で1時間、適切な大きさのガラスバイアル中で洗浄する。 PFAを取り除くために検体をナレーターで静かに揺する。溶液を交換する際には、試料に損傷を与えないでください。 </li><li> 1％（w / v）のオルト過ヨウ素酸を調製するには、100mLのDW中に1gのオルト過ヨウ素酸を溶解する。 </li><li>内因性ペルオキシダーゼ反応を防ぐために、1％オルト過ヨウ素酸中で20分間RTで検体をインキュベートする。 </li><li> 0.004gのパパインを0.025mol / Lのトリス - 塩酸緩衝液（pH7.6）100mLに溶解して0.004％（w / v）のパパインを調製する。 </li><li> DWで10分間RTで検体を洗浄する。 </li><li>穏やかに振盪しながら恒温槽中で37℃で2時間、新たに調製した0.004％パパインで検体をインキュベートする。 </li><li> Dを用いて検体を洗浄するWで50〜60分間RTでインキュベートした。 </li><li>試料を4％CO / Nで4％PFAに保存する。 </li></ol></li><li> 2日目：酵素処理 <ol><li>保管した試験片をDWで4時間、室温で1時間洗浄する。 </li><li>穏やかに振盪しながら恒温槽中で37℃で2時間、新たに調製した0.004％パパインで検体をインキュベートする。 </li><li> DWで50〜60分間RTで標本を洗浄する。試料を4％CO / Nで4％PFAに保存する。 </li></ol></li><li> 3日目：凍結処理 <ol><li>上記のように、保存した試験片を室温で1時間、DW 4xで洗浄する。 </li><li> 2.5g、5gおよび10gのスクロースをそれぞれ0.01M PBS（100mLのPBS中に溶解することにより2.5％（w / v）、5％（w / v）および10％ pH7.4）。 </li><li> 2.5％（w / v）、5％（w / v）および10％（w / v）スクロースにそれぞれ30分間浸漬する。 </li><li>試料を-20℃で30〜60分間凍結し、RTで完全に解凍する。サイクルを3回繰り返す。 </li><li>上2％Triton X-100（v / v）を補充し、2mLのTriton X-100を100mLの0.01M PBS（pH 7.4）に加える。 </li><li> 4％CO / Nで2％Triton X-100に検体を保存する。 </li></ol></li><li> 4日目：一次抗体インキュベーション <ol><li>ウシ血清アルブミン（BSA）0.2g、トリトンX-100 0.3ml、アジ化ナトリウム0.1gを0.01M PBS（pH 7.4）100mlに溶解して、一次抗体の希釈液を調製する。 注意：アジ化ナトリウムは急性毒性があります。吸入および接触は避けなければならない。適切なPPEを着用してください。 </li><li>上記の希釈バッファー（ステップ3.4.1）で1：600の一次抗体（NFP）を希釈する。検体をNFP抗体と4℃で3〜6日間インキュベートし、検体をナテーター上で静かに揺り動かします。より大きな検体は、 インキュベーション時間の増加を必要とする。 </li></ol></li><li> 二次抗体インキュベーション <ol><li>未結合の一次抗体を除去するために、RTでそれぞれ1時間、PBS 4x中で検体を洗浄する。 </li&gt; <li> 0.2gのBSAと0.3mLのTriton X-100を100mLの0.01M PBS（pH 7.4）に溶解することにより、二次抗体のための希釈溶液を調製する。 </li><li>希釈バッファー（ステップ3.5.2）で二次抗体（ペルオキシダーゼ結合、アフィニティー精製ヒツジ抗マウスIgG-HRP）を1：600希釈する。検体を2次抗体と4℃で3日間インキュベートし、検体をナイトレーター上で静かに揺り動かします。 </li></ol></li><li> 着色 <ol><li>溶液を暗所に保ち、ヒューム皿の下で0.05 mol / L Tris-HCl緩衝液（pH 7.6）100 mLに3,3&#39;-ジアミノベンジジンテトラヒドロクロリド（DAB）0.002 gを加えてDAB着色溶液を調製する。 </li><li> PBSで4℃で1時間、標本を室温で洗浄する。 </li><li>新たに調製したDAB着色溶液に30％H 2 O 210μLを添加し、4℃/ Nでまたは3日間インキュベートする。 </li><li>反応をplaで停止する最適染色強度に達したときに、0.04％アジ化ナトリウムを含むPBS中で標本を染色する。 注：西洋ワサビペルオキシダーゼを阻害するので、このステップまでアジ化ナトリウムを使用しないでください。 </li></ol></li><li> 染色組織のイメージング <ol><li>慎重に、検体をPBSを含むペトリ皿（高さ5cm、直径10cm）に移す。実体顕微鏡に取り付けられたカメラを使用して、全マウント画像をキャプチャします。 注：完全にマウントされた組織は、透明なガラス皿上のPBSに完全に浸した状態で撮像する必要があります。 </li></ol></li></ol>

<ol>
	<li>Kuratani, S., Tanaka, S., Ishikawa, Y., Zukeran, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+hypoglossal+nerve+in+the+chick+embryo+as+observed+by+the+whole-mount+nerve+staining+method.">Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method.</a> Am. J. Anat. 182 (2), 155-168 (1988).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+the+avian+vagus+nerve+with+special+reference+to+the+morphological+innervation+of+heart+and+lung.">Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.</a> Anat. Embryol. (Berl). 182 (5), 435-445 (1990).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+avian+trigeminal+nerves.">Peripheral development of avian trigeminal nerves.</a> Am. J. Anat. 187 (1), 65-80 (1990).</li><li>Yi, S. Q., 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=House+musk+shrew,+Suncus+murinus:+a+novel+and+natural+obesity-resistant+animal+model.">House musk shrew, Suncus murinus: a novel and natural obesity-resistant animal model.</a> Obes. Metab. 6, 22-28 (2010).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+innervation+of+the+gallbladder+in+humans+and+Suncus+murinus+with+special+reference+to+morphological+understanding+of+gallstone+formation+after+gastrectomy.">Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.</a> World J. Gastroenterol. 13 (14), 2066-2071 (2007).</li><li>Yi, S. Q., 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=Anatomical+study+of+the+pancreas+in+the+house+musk+shrew,+Suncus+murinus,+with+special+reference+to+the+blood+supply+and+innervation.">Anatomical study of the pancreas in the house musk shrew, Suncus murinus, with special reference to the blood supply and innervation.</a> Anat. Rec. A Discov. Mol. Cell Evol. Biol. 273 (1), 630-635 (2003).</li><li>Yi, S. 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Q., 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=Surgical+anatomy+of+the+innervation+of+the+pylorus+in+humans+and+Suncus+murinus,+in+relation+to+the+surgical+technique+for+pylorus-preserving+pancreatoduodenectomy.">Surgical anatomy of the innervation of the pylorus in humans and Suncus murinus, in relation to the surgical technique for pylorus-preserving pancreatoduodenectomy.</a> World J. Gastroenterol. 12 (14), 2209-2216 (2006).</li><li>Yi, S. Q., Ren, K., Kinoshita, M., Takano, N., Itoh, M., Ozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+Extrahepatic+Biliary+Tract,+With+Special+Reference+to+the+Direct+Bidirectional+Neural+Connections+of+the+Gall+Bladder,+Sphincter+of+Oddi+and+Duodenum+in+Suncus+murinus,+in+Whole-Mount+Immunohistochemical+Study.">Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.</a> Anat. Histol. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+nerve+cells+from+the+duodenum+to+the+sphincter+of+Oddi+and+gallbladder+of+the+Australian+possum.">Projections of nerve cells from the duodenum to the sphincter of Oddi and gallbladder of the Australian possum.</a> Gastroenterology. 104 (1), 130-136 (1993).</li><li>White, J. J., Reeber, S. L., Hawkes, R., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wholemount+immunohistochemistry+for+revealing+complex+brain+topography.">Wholemount immunohistochemistry for revealing complex brain topography.</a> J. Vis. Exp. (62), e4042 (2012).</li></ol>

著者らは、利益相反を宣言していない。

この研究は、ニューロフィラメントタンパク質に対する抗体を用いて肝外胆道の神経支配を視覚化するための実験手順を説明している。このプロトコルは、KurataniおよびTanaka 1,2,3によって記述されたプロトコルから適合させた。 この実験では、全マウント染色法が2〜3週間続くので、各実験のタイムラインを計画します。組織を入れた容器は、凍結/解凍プロセスを除いて、常にナットで回転させる必要があります。特に、一次抗体および二次抗体とのインキュベーション中、試験片を反応液に均一に曝露するために、検体をナッターで回転させながら冷蔵保存室にセットした。 1％（w / v）オルト過ヨウ素酸、0.004％（w / v）パパイン、および一次/二次抗生物質のための希釈バッファーダイスは各実験ごとに新しく作らなければならない。プロトコール全体を通して、溶液を交換する際に試料に触れたり損傷したりしないように、溶液は鉗子15で取り除かれる代わりに注ぎ出されるべきである。 PFAで固定した後、サンプルをDWまたはPBSで十分に洗浄する必要があります。さらに、パパインインキュベーションによる酵素処理は重要である。抗原回収に使用される熱および酵素は、抗体の浸透のために組織を調製するのに役立つ。溶液の浸透を促進するために、-20℃で30分間または-80℃/ 15分間の凍結処理によって、組織の外側膜を破壊しなければならない。 溶液が完全な検体をインキュベーションできることを保証するために、十分な量の緩衝液に実験検体を完全に浸すことが重要です。一次抗体のインキュベーション時間は、経験的に決定しなければならないdを異なる組織およびサンプルサイズについて測定した。通常、2〜3cmのサンプルでは3日でなければなりません。最適な希釈は、各抗体について経験的に決定されなければならない。適切な抗体バッファーを使用する場合は、1：600の希釈液を1次抗体と2次抗体の両方に推奨します。 この研究は、 S. murinusの胆道における神経フィラメントタンパク質発現を明らかにする多目的全マウント免疫組織化学的アプローチのプロトコルを詳細に記載している。この方法は、多くの種のすべての内臓器官の神経支配を分析するために使用することができます。さらに、このプロトコールは、他のニューロン抗体を試験するために適合させることもできるが、抗体の最適化を行うべきである。 しかしながら、この技術は、浅い神経組織を標識し、3次元の神経支配モデルを構築することには限界があった。また、神経線維の特徴（ すなわち交感神経または同位体）を同定することができなかった無関心、運動または感覚など ）。

家のムスクシュル、 Suncus murinusは、InsectivoraとSoricidae目に属する。この小さな哺乳類は東南アジア全域に分布しており、人間の住居や牛のペンの近くにある家や草地に生息しています4 。この種は、マウス、ラット、ウサギのような他の実験動物と比べてヒトに類似した一般的な形態学的特徴を示す。以前、膵臓6 、大十二指腸乳頭7 、幽門8 、胆嚢5 、および肝外胆道9の末梢神経を標識するために、 S.ムリナスニューロフィラメントタンパク質（NFP）に対する末梢ニューロンマーカーを用いた全マウント免疫染色を用いた。 NFPは、成熟した神経細胞骨格の一部である巨大分子複合体である。神経フィラメント関連タンパク質NFPとザイソソームとの間の相互作用を媒介する。酵素機能およびリンカータンパク質の構造はいずれもNFP 10によって調節される。 NFP複合体は、NF-L（70kDa）、NF-M（150-160kDa）、およびNF-H（200kDa）の3つのポリペプチドからなる。 NFPは、中枢神経系および末梢神経系の両方におけるニューロンの軸索に見出され得る。抗ヒトNFP抗体は、中枢神経系および末梢神経系の軸索を標識することが示されている。解剖学的および電気生理学的研究により、括約筋、胆嚢および近位胃腸管が11,12,13,14に接続されていることが示された。しかしながら、それらのニューロン結合の形態学的特徴はまだ定義されていない。 この研究は、 S. murinus bilの神経支配を標識するための全マウント免疫組織化学的染色法の使用を実証しているNFP抗体を用いたiary tract。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">orthoperiodic acid</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">162-00732</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">papain</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">164-00172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">010-15131</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium azide</td>
			<td style="width:71px;">Nacalai Tesque</td>
			<td style="width:119px;">312-33</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neurofilament protein (NFP) antibody</td>
			<td style="width:71px;">Dako</td>
			<td style="width:119px;">M0762, lot 089, clone: 2F11</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td>
			<td style="width:71px;">MBL</td>
			<td style="width:119px;">Code 330</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">349-00903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I-0125</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

この研究は、 Suncus murinus（ S. murinus）における胆道の神経支配を標識するためにニューロフィラメントタンパク質抗体を使用して、全マウント免疫組織化学染色方法を詳細に説明する&#160; ）。最初に、検体をS. murinusから解剖し、4％パラホルムアルデヒド（PFA）で固定した。第2に、酵素処理および潜在的な内因性ペルオキシダーゼ不活性化を行った。次いで、試験片を一次抗体である抗神経フィラメントタンパク質抗体に3-6日間曝露した。その後、西洋ワサビペルオキシダーゼと結合した二次抗体とインキュベートした。呈色反応は、検体を3,3&#39;-ジアミノベンジジン（DAB）基質と反応させることによって明らかにした。この方法は、すべての内臓器官の神経支配を分析するために適用することができる。さらに、このプロトコールは、他のニューロン抗体を試験するために適合させることもできるが、抗体の最適化最初に行う必要があります。この方法はもともとKuratani and Tanaka 1,2,3によって導入された。

図1〜4は、 S. murinusの肝外胆管におけるNFP陽性神経線維の典型的な結果を示す。 （ 図1 ）、胆嚢（ 図2 ）、上部胆管（ 図3 ）、および十二指腸乳頭領域（ 図4 ）の画像全体において、NFPに対する抗体は高い特異性および最小限度を再現可能に標識したバックグラウンド。 形状および大きさにかかわらず、標本のすべての組織について、被験者の神経を同時に染色することができる。肝外胆管の神経支配に加えて、食道および胃の迷走神経の走りおよび分布密度は明白に示された（ 図1 ）。 図2 ）。 上部胆管には、2種類の神経束が標識されていた。細かい神経束は、不規則で密集した神経回路網を形成し、接着して走り、胆道の上/中に存在した。より厚い神経束は胆道の表面に平行に分布していた（ 図3 ）。 総胆管には青色のネオプレンラテックス、白いネオプレンのラテックスにはラベリングした。総胆管の末端および十二指腸乳頭領域の細い神経線維は、高cで明瞭に示された<html> （ 図4 ）。 <img alt="図1" class="xfigimg" src="/files/ftp_upload/55483/55483fig1.jpg" /> 図1：胆道、食道、および胃におけるNFP陽性神経線維。矢印は食道に沿って胃に流れている迷走神経を示しています。 CBD、共通胆管;食道、食道; St、胃。スケールバー=1300μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig1large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a> <img alt="図2" class="xfigimg" src="/files/ftp_upload/55483/55483fig2.jpg" /> 図2：胆嚢のNFP陽性神経線維。胆嚢の血管は白いラテックスで標識した。豊富な神経支配が胆嚢の頸部に生じた（矢印）。スケールバー=1,000μm。ref = &quot;https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig2large.jpg&quot; target = &quot;_ blank&quot;&gt;この図の拡大版を見るには、ここをクリックしてください。 <img alt="図3" class="xfigimg" src="/files/ftp_upload/55483/55483fig3.jpg" /> 図3：上胆管のNFP陽性神経線維。 2種類の神経束が観察された。 1つのタイプは、接着して走り、肝外胆道の上/中に存在する細い神経束であった（矢印）。他のタイプは、肝外胆道の表面に平行に分布し、胆嚢と十二指腸（三角）の間を走るより厚い神経束であった。 PV、Portal Vein。スケールバー=650μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig3large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a> <img alt="図4" class= &quot;xfigimg&quot; src = &quot;/ files / ftp_upload / 55483 / 55483fig4.jpg&quot; /&gt; 図4：十二指腸乳頭領域におけるNFP陽性神経線維。総胆管は青色ラテックスで標識し、血管は白いラテックス（*）で標識した。矢印は総胆管の末端の神経支配を示し、三角は総胆管および血管から来る十二指腸乳頭領域（P）の神経支配を示す。スケールバー=1,000μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig4large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a>

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Suncus murinusの肝外胆管における神経フィラメントタンパク質の発現を視覚化するための全身免疫組織化学的アプローチ。ここに表示されます。このプロトコルは、 S. murinusまたは他の種のすべての内臓器官の神経支配を分析するために使用することができる。

全身免疫組織化学的方法を用いて胆道の内腔を調べる<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

using-whole-mount-immunohistochemical-method-to-study-innervation

Nanyang Technological University

Research

JoVE Journal

Biology

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K ビュー.  Tokyo Metropolitan University.  Suncus murinusの肝外胆管における神経フィラメントタンパク質の発現を視覚化するための全身免疫組織化学的アプローチ。ここに表示されます。このプロトコルは、 S. murinusまたは他の種のすべての内臓器官の神経支配を分析するために使用することができる。 

ビデオ: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

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

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

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

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

Method for Whole Mount Antibody Staining in Chick

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 ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

ハイC：ゲノムの三次元構造を研究する方法。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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

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

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

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

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

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

Whole-mount 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 ...

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

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

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

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

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

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

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

ハイスループットその場で</em胎児マウス下部尿生殖路のmRNAの発現パターンを特徴づける>ハイブリダイゼーション法

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...