Name: immunostaining to visualize murine enteric nervous system development
Uploaded: 2015-04-29T13:00:00
Description: Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

この作品はアメリカの小児外科協会財団賞（AG）と健康K08DK098271（AG）の国立研究所メートがサポートされていました。

ウィスコンシン大学の動物実験委員会は、すべての手順を承認しました。 ソリューションの調製<ol><li>解剖緩衝液およびリンス液として1×リン酸緩衝生理食塩水（PBS）を使用します。 </li><li>ボトルにスクロースおよび場所の30グラムを計量することにより、30％スクロースを準備します。 1×PBS 99 mlを加え、1mlの10％アジ化ナトリウムを追加します。ショ糖の全てが?...

<ol>
	<li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+determinants+of+the+independence+and+complexity+of+the+enteric+nervous+system.">Developmental determinants of the independence and complexity of the enteric nervous system.</a> Trends Neurosci. 33 (10), 446-456 (2010).</li><li>Amiel, J., Sproat-Emison, E., 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=Hirschsprung+disease,+associated+syndromes+and+genetics:+a+review.">Hirschsprung disease, associated syndromes and genetics: a review.</a> J Med Genet. 45 (1), 1-14 (2008).</li><li>Erickson, C. S., Barlow, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+and+polyvalent+antiserum+to+chicken+choline+acetyltransferase.">Monoclonal antibodies and polyvalent antiserum to chicken choline acetyltransferase.</a> J Neurochem. 46 (3), 968-976 (1986).</li><li>Tooyama, I., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+encoded+by+an+alternative+splice+variant+of+choline+acetyltransferase+mRNA+is+localized+preferentially+in+peripheral+nerve+cells+and+fibers.">A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.</a> J Chem Neuroanat. 17 (4), 217-226 (2000).</li><li>Koga, T., Bellier, J. -. P., Kimura, H., Tooyama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+Choline+Acetyltransferase+of+Peripheral-Type+(pChAT)+in+the+Trigeminal+Ganglion+Neurons+of+the+Non-Human+Primate+Macaca+fascicularis.">Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis.</a> Acta histochemica et cytochemica. 46 (2), 59-64 (2013).</li><li>Sang, Q., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+chemical+coding+of+cholinergic+neurons+in+the+small+and+large+intestine+of+the+mouse.">The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse.</a> Anat Rec. 251 (2), 185-199 (1998).</li><li>Lei, J., Howard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+Hand2+in+enteric+neural+precursor+cells+affects+its+functions+in+neurogenesis,+neurotransmitter+specification+and+gangliogenesis,+causing+functional+aganglionosis.">Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis.</a> Development (Cambridge, England). 138 (21), 4789-4800 (2011).</li><li>Erickson, C. S., Lee, S. J., Barlow-Anacker, A. J., Druckenbrod, N. R., Epstein, M. L., Gosain, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+cholinergic+myenteric+neurons+during+enteric+nervous+system+development:+comparison+of+different+ChAT+fluorescent+mouse+reporter+lines.">Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.</a> Neurogastroenterol Motil. 26 (6), 874-884 (2014).</li><li>Teitelbaum, D. H., Caniano, D. A., Qualman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+Hirschsprung's-associated+enterocolitis:+importance+of+histologic+correlates.">The pathophysiology of Hirschsprung's-associated enterocolitis: importance of histologic correlates.</a> J Pediatr Surg. 24 (12), 1271-1277 (1989).</li><li>Aslam, A., Spicer, R. D., Corfield, 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=Children+with+Hirschsprung's+disease+have+an+abnormal+colonic+mucus+defensive+barrier+independent+of+the+bowel+innervation+status.">Children with Hirschsprung's disease have an abnormal colonic mucus defensive barrier independent of the bowel innervation status.</a> J Pediatr Surg. 32 (8), 1206-1210 (1997).</li><li>Puig, I., Champeval, D., De Santa Barbara, P., Jaubert, F., Lyonnet, S., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Pten+in+the+mouse+enteric+nervous+system+induces+ganglioneuromatosis+and+mimics+intestinal+pseudoobstruction.">Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction.</a> J Clin Invest. 119 (12), 3586-3596 (2009).</li></ol>

私たちの研究室等はHSCR腸内の欠陥があっても神経節の小腸5,15,16に、無神経節結腸に限らず、近位に延長されていないことを示しています。これらの変化はENS神経密度と神経伝達物質表現型の変化を含み、HSCRを有する患者において観察された運動障害を説明することができます。私たちは、ENS形成の決定要因を理解するための努力で上記の技術を利用しています。具体的には、これ?...

運動、栄養吸収、およびローカルの血流を制御する機能腸管神経系（ENS）は、生命1に不可欠です。 ENSは、それらはニューロンおよびグリア細胞を含む神経節に分化腸を移動し、コロニー形成、増殖、神経堤細胞（NCC）によって形成されます。ヒルシュスプルング病（HSCR、男性でのオンラインメンデル遺伝）、1 4,000で出生の発生率とmultigeneic先天性障害は、破壊されたENSの形成を研究するためのプロトタイプの病気と考えることができます。 HSCRでは、NCCはに移動し、遠位後腸2の変数の長さを植民地化することができません。また、他の一般的な胃腸（GI）は、肛門直腸奇形、腸atresias、および運動障害などの小児集団における発達障害は、基本的なENS機能の障害と関連しており、おそらく微妙な、過小評価、解剖学的変化とで機能的変化と関連していますENS 3-6。そのため、私たちはENS形成の発達決定要因を理解することを可能にする技術は、病因および小児消化管障害の治療の可能性に光を当てることができます。 移行と植民地化の後、NCCは、それらの神経伝達物質の表現型に特異的なマーカーでニューロンに分化します。コリン作動性ニューロンは、腸溶性ニューロン7の約60％を含み、かつコリンアセチルトランスフェラーゼ（チャット）、興奮性神経伝達物質アセチルコリンのための合成酵素を染色することによって検出することができます。歴史的には、コリン作動性ニューロンを可視化する試みは、中枢神経系に対して向けられた抗体の抗原特異性（CNS）は、末梢神経系（PNS）のChAT 8-10に対してチャット異なるによって混乱しました。しかし、胎盤のChATに対する抗体は、両方の中央と周辺のChAT 11-13を認識し、我々は最近、visuaを可能にする技術が記載されています高感度のENSのコリン作動性ニューロンのlization以前の開発でチャットレポーター線14で達成されたよりも。 ここでは、ニューロンにおけるENSの神経伝達物質の発現を可視化するマウス胚GI管の、固定および免疫染色を解剖するための手法を提示します。これらの研究のために、我々は、 チャット-Creマウスを利用しているR26Rと交配：floxSTOP：チャットのCreを生成するためにtdTomato動物を、R26R：floxSTOP：tdTomatoマウス （原稿全体のChAT-CreをtdTomatoと定義されます）。これらの動物は、その後のChAT発現14を検出する蛍光レポーターの両方を発現するマウスを得るために、ホモ接合のChAT-GFPレポーターマウスと交配させました。これら二つのレポーター動物は、C57BL / 6Jバックグラウンド上にあり、（ジャクソン·ラボラトリーズ、バーハーバー、ME）は、市販されています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

我々は以前のChAT発現14を検出GFPおよびtdTomato蛍光レポーターの両方を発現するマウスの生成を記載しています。簡単に説明すると、 チャットCreマウスは、R26Rと交配させた：floxSTOP：チャットのCreを生成するためにtdTomato動物; R26R：floxSTOP：（チャット-CreをtdTomatoと呼ばれる）tdTomatoマウスを。これらの動物は、その後、ホモ接合のChAT-GFPレ...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

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. 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 ...

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