Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
Uploaded: 2017-06-15T12:30:00.000+00:00
Duration: 7 min 23 s
Description: 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

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. 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+major+duodenal+papilla+in+humans+and+Suncus+murinus,+from+the+perspective+of+preserving+innervation+in+organ-saving+procedures.">Surgical anatomy of the innervation of the major duodenal papilla in humans and Suncus murinus, from the perspective of preserving innervation in organ-saving procedures.</a> Pancreas. 30 (3), 211-217 (2005).</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+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. Embryol. 45 (3), 184-188 (2016).</li><li>Wang, H., 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=Neurofilament+proteins+in+axonal+regeneration+and+neurodegenerative+diseases.">Neurofilament proteins in axonal regeneration and neurodegenerative diseases.</a> Neural Regen. Res. 7 (8), 620-626 (2012).</li><li>Wyatt, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+the+sphincter+of+Oddi+to+the+stomach,+duodenum+and+gall-bladder.">The relationship of the sphincter of Oddi to the stomach, duodenum and gall-bladder.</a> J. Physiol. 193 (2), 225-243 (1967).</li><li>Mawe, G. M., Kennedy, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+neurons+provide+nicotinic+fast+synaptic+input+to+sphincter+of+Oddi+neurons+in+guinea+pig.">Duodenal neurons provide nicotinic fast synaptic input to sphincter of Oddi neurons in guinea pig.</a> Am. J. Physiol. 277, G226-G234 (1999).</li><li>Seo, J. H., Cho, S. S., Lee, I. S., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+neuropeptidergic+properties+of+the+duodenal+neurons+projecting+to+the+gallbladder+in+the+golden+hamster.">Anatomical and neuropeptidergic properties of the duodenal neurons projecting to the gallbladder in the golden hamster.</a> Arch. Histol. Cytol. 65 (4), 317-321 (2002).</li><li>Padbury, R. T., Furness, J. B., Baker, R. A., Toouli, J., Messenger, J. 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...

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><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>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?...

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

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

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

マウス腸神経系の開発を可視化するために免疫染色

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

発生生物学

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

がん研究における治療・診断抗体生体分布の評価のための生体内免疫蛍光局所法

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

がん研究

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

マウス肝臓における胆管密度の決定

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

腸神経系のその場のCa 2+イメージングに

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

神経科学

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

マウスにおけるカハール（ICC）ネットワークの間質細胞の可視化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

生物学

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

神経化学的マーカーのためのビオシチンで満たされたと加工セクションの免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

マウス小腸における上皮内リンパ球の分体イメージング

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

免疫・感染学

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

Source: Michael S. Lee1 and Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots &#34;immuno-,&#34; in reference to antibodies, and &#34;histo-,&#34; in reference to the tissue sections used in IHC. The root &#34;cyto-&#34; in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably.
In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specific binding of the tagged antibody to the antigen. In the case of IHC, thin slices of tissue are immobilized on a slide to maintain the structure of the tissue before being stained, allowing the visualization of antigens in the context of whole tissues (Figure 1). In the case of ICC, cells are distributed evenly on a slide before being stained, allowing the visualization of antigen distribution within individual cells but not within the structure of any specific tissue. Due to the similarities between the two protocols, this protocol will focus on IHC to address the additional complexities of sample preparation involved in IHC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10498/10498fig1.jpg" /> 
Figure 1: Outline of IHC Protocol. Visual outline of an IHC protocol for paraffin-embedded tissue dissected from a mouse. This protocol uses a biotinylated secondary antibody and strepavidin-HRP to visualize the location of antibody binding. Other options, such as fluorescently tagged antibodies, are also possible. <a href="https://www.jove.com/files/ftp_upload/10498/10498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first major decision when performing IHC is how to prepare the tissue sections in order to maintain the structure of the tissue throughout the staining process. The two main choices are formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. There is no simple answer as to which method to use as it depends on what downstream analysis will be conducted. Formalin-fixation of paraffin embedded tissues is generally thought to better preserve tissue morphology for optimal imaging while freezing fresh tissue can preserve protein function for subsequent assays outside of IHC. In addition, fresh frozen tissue sections have been shown to be more suitable for gene expression analysis (2). A third consideration is whether or not the antibodies for your antigen of interest are suitable for either fixed or frozen tissue sections, as some antibodies have only been optimized for a specific type of section and may not work for others. Finally, one also needs to determine how long they need to store the tissue sections, as fresh frozen samples must be kept at -80&#176;C and may not last beyond one year while fixed sections can be stored for much longer at room temperature. These are a few of the major considerations for determining whether to use formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. Ultimately, if one has enough tissue, it may be best just to have some of both.
In this experiment, we set out to determine if cyclin D1 expression was increased in enlarged spleens from a spontaneous mouse model of lymphoma development. Splenic tissue samples were first isolated from either wild-type mice, transgenic mice that do not have lymphoma, or transgenic mice that have spontaneously developed lymphoma. The spleen tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using a mouse anti-cyclin D1 primary antibody followed by a horse anti-mouse secondary antibody, and developed using 3,3-diaminobenzidine (DAB). The sections were then counterstained in Harris Hematoxylin Solution and then the sections were imaged at 20X magnification.
Reagents
Paraffin-Embedded Sections
<ol type="a">
	<li>4% Paraformaldehyde (PFA)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100%, 95%, 80%, 75%, and 50%). Can be diluted from 100% stock using double distilled water (ddH2O)</li>
	<li>Xylene</li>
	<li>IHC compatible glass slide to ensure tissue section remains attached throughout the entire procedure. IHC compatible glass slides have a specialized coating and are readily available from multiple retailers. If performing ICC, use a chambered slide. Chambered slides allow for cells to be seeded in the chambers and placed in the incubator until cells attach to the slide and reach proper confluency, at which point the chambers can be removed and staining can proceed in the same manner as IHC.</li>
	<li>Paraffin</li>
	<li>0.3% Hydrogen peroxide (H2O2)/methanol: To prepare, add 1 mL 30% H2O2 to 99 mL methanol. Store at -20&#176;C</li>
	<li>Antigen retrieval buffer: IHC citrate buffer pH 6.0</li>
</ol>
Fresh Frozen Sections
<ol type="a">
	<li>Optimal cutting temperature (OCT) embedding compound</li>
	<li>Optimal fixative: 4% PFA or acetone that has been cooled to -20&#176;C</li>
</ol>
Staining
<ol type="a">
	<li>Blocking buffer: Should be determined by user. One example is horse serum diluted in 1X PBS</li>
	<li>Diluted primary antibody: see manufacturer specifications</li>
	<li>Diluted biotinylated secondary antibody: see manufacturer specifications</li>
	<li>Diluted avidin-Horseradish Peroxidase (HRP): Only for peroxidase visualization. See manufacturer specifications.</li>
	<li>DAB or another compatible substrate</li>
	<li>Counterstain (optional)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100% and 95%)</li>
	<li>Xylene</li>
	<li>Organo/Limonene Mount</li>
</ol>

Immunohistochemistry &amp; Immunocytochemistry: Tissue Imaging

免疫組織化学と免疫細胞化学:光顕微鏡による組織イメージング

Source: Michael S. Lee1 and Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and ...

Education

JoVE Science Education

高等生物学

免疫学

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. <a href="https://app.jove.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Following anesthetization as described in the text protocol, prepare for the tail vein inoculation of the antibody solutions. Disinfect the tail of the animal by wiping three times with an alcohol wipe. Dilate the tail veins by warming with a heat pad for approximately 30 seconds; avoid heating the entire animal. Using a 27-gauge tail vane catheter insert the butterfly needle into one of the two lateral tail veins.
Visualize blood backflow into the catheter to ensure that the needle is properly placed so that the solutions will fully enter the animal versus being captured in the tail.
Use a piece of surgical tape to carefully fix the tail with the inserted needle to the stage. Flush the catheter with 25 microliters of sterile phosphate-buffered saline. Then, inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 microliters of sterile PBS. Remove the needle from the tail, and apply pressure to stop any bleeding.
Perform humane euthanasia of the mouse, as described in the text protocol, and lay the mouse in the supine position. Use surgical scissors and forceps to excise tumor tissues. Use forceps to grasp only the outer layer of skin between the set of mammary glands closest to the tail, and to make a small incision with a pair of surgical scissors. Introduce the closed scissors into the cut, and slowly open the tip to carefully separate the skin from the underlying abdominal wall membrane, keeping it intact.
Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the third and fourth mammary glands, make a horizontal cut across the abdomen, to allow retraction of the skin and visualization of the mammary glands. Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors.
Place excised tissues into tissue disposable base molds that are pre-labeled and filled with optimal cutting temperature embedding medium. Quickly freeze the molds by placement on dry ice. In order to study off-target delivery, excise other tissues or organs of interest. Using a cryostat, section frozen tissue blocks at 10-micron thickness, and place adjacent sections onto pre-labeled adhesion glass slides.
Rinse frozen tissue slides with room temperature PBS for 5 minutes to remove the embedding medium. Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining. Fix the tissue sections with 4% paraformaldehyde solution for 5 minutes. After rinsing the slides in PBS for 5 minutes, permeabilize tissue sections with 0.5% Triton X-100 in PBS for 15 minutes. Rinse the slides again in PBS for 5 minutes.
Block the tissues with PBS containing 3% weight per volume bovine serum albumin and 5% volume per volume goat serum for 1 hour at room temperature. After rinsing the slides in PBS for 5 minutes, incubate the sections with record-keeping primary antibodies, such as a common nuclear, vascular, or cytoplasmic marker. Here, rat anti-mouse CD31 is used at a 1 to 100 dilution in the blocking solution. The slides with primary antibody are left overnight at 4 degrees Celsius, protected from dehydration on a slide tray.
Following incubation, rinse the slides in PBS for 5 minutes three times. Change the PBS each time. Incubate the slides with secondary antibodies to label the primary antibodies. For this application, visualize anti-B7-H3 antibody using AlexaFluor-546 conjugated goat anti-rabbit antibody, and visualize CD31 with AlexaFluor-488 goat anti-rat secondary antibody in blocking solution. Protect the slides from light and dehydration on a slide tray for 1 hour at room temperature.
After rinsing the slides in PBS, as before, apply one drop of the mounting medium into the center of the tissue slice. Carefully place a coverslip avoiding entrapment of air bubbles. Seal the edges of the coverslip with clear nail polish and allow to dry. Proceed with confocal microscopy imaging and quantitative image analysis, as described in the text protocol.

全身免疫組織化学的方法を用いて胆道の内腔を調べる

要約

さらに動画を探す

この動画の章