这项工作是支持的美国bythe小儿外科协会基金会奖（AG）和健康K08DK098271全国学院（AG）。

威斯康星州的动物护理和使用委员会的批准大学的所有程序。 1.准备解决方案<ol><li>使用1×磷酸盐缓冲盐水（PBS），为解剖缓冲液和冲洗液。 </li><li>由30克蔗糖和地点到瓶中制备30％的蔗糖。添加99毫升1×PBS中并加入1毫升10％的叠氮化钠。调匀，直到所有的蔗糖溶解。直到需要保存在4℃。 </li><li>制备通过混合的1×PBS，3％牛血清白蛋白（BSA）和0.1％的Triton-X-100的封闭溶液。调匀，直到需要在冰箱里储存。 </li><li>制备在1×PBS中的8％的多聚甲醛（PFA）溶液通过称重的PFA的适量的1×PBS中，然后孵育在65℃直到它完全溶解。商店在25毫升等份在-20℃冷冻，直到需要。稀释至4％PFA中的1×PBS在使用的当天。 </li></ol> 2.胚胎和肠道解剖离子<ol><li>按照机构动物护理和使用委员会批准的方案，安乐死定时怀孕小鼠和子宫转移到60毫米培养皿中含有冰冷的1×PBS。 </li><li>在使用锋利的剪刀解剖镜下，切开子宫壁并暴露出胚胎。除去从子宫和地点胚胎成含冰冷的1×PBS中的清洁60毫米培养皿。 </li><li>安乐死在冰冷的1X PBS每个胚胎被斩首。如果您使用的是含有荧光蛋白转基因小鼠，在荧光照明，标识正转基因胚胎。 </li><li>解剖使用解剖显微镜每个胚GI道。用细镊子，定向，使得所述左侧朝上，右侧是针对培养皿底部的胚胎。从胚胎中取出上体壁以暴露内部器官。将背体壁和内脏之间的细镊子。铬oss骨粉对对方的镊子在剪刀状切割动作，从胚胎取出内脏。 </li><li>从周围器官进一步分解剖GI道远，然后将每个GI道到含有冰冷的1×PBS 1.5ml微量离心管中。 </li></ol> 3.固定GI大片<ol><li>冲洗每个GI道3次，用冰冷的1×PBS，然后替换用4％PFA。固定在摇摆平台在RT 1.5小时在1.5 ml微量离心管在胃肠道。冲洗胃肠道3次，每次5分钟，RT，然后进行1小时的摇动平台上。在此阶段，存储在胃肠道，在4℃下在30％蔗糖中含有1x PBS中的0.1％的叠氮化钠，直到需要。 注意：可替换地，存储在30％蔗糖的胚胎为长达一年而不对组织的完整性的任何影响。在30％的蔗糖样品的存储允许的样品无论是对免疫染色或进入十月为低温sectioni的购买处理NG。另外，继续进行下面详细介绍的免疫协议。 </li></ol> 4.免疫染色协议<ol><li>如果样本已储存在30％的蔗糖，冲洗他们在摇摆平台上在1×PBS中3次20分钟。 </li><li>放置在胃肠道进入封闭溶液在摇摆平台上1小时，在室温。 </li><li>除去封闭液孵育胃肠道与稀释于封闭为任一4小时，在室温或O / N在4℃下在摇摆平台上的解决方案的主要抗体的适当的量。使用1：1000稀释的人抗胡抗体（血清自患者获得），1：1000稀释的鸡抗 - 绿色荧光蛋白（GFP）的抗体和1：100稀释的山羊抗胆碱抗体。 注意：我们利用了从患者局部获得的人抗胡抗体，然而，抗胡抗体是可商购的，例如，小鼠抗胡，（使用1：500稀释）。 </li><li>在冲洗1X PBS胃肠道3次，每次5分钟，然后进行1小时，在室温在摇摆平台上。 </li><li>在封闭溶液在摇摆平台上为4小时，在室温或O / N在4℃下500：取代的1×PBS稀释1的二次抗体。使用驴抗人胺反应性染料如Dylight，驴抗鸡的Cy2和驴抗山羊Cy5的。如果使用小鼠抗胡抗体然后用1:500稀释的驴抗小鼠胺反应性染料405。 注：内源性GFP和tdTomato表达和免疫染色随发育年龄的清晰度的强度。我们通常免疫染色胚胎胆单独在0.2ml管中加入150μl的染色溶液，以减少所使用的抗体的量，同时确保组织的有效染色。 </li><li>冲洗在1×PBS中的3倍胃肠道5分钟，然后进行1小时，在室温在摇摆平台上。 </li><li>放置几滴荧光封固剂用DAPI的载玻片上。沉浸GI TRA克拉到荧光封固剂并直接在组织上添加一个玻璃盖。荧光封固剂-G是一种水溶性化合物，它提供了半永久性密封。 注：使用荧光安装介质如的Vectashield这是基于安装介质，防止褪色和抗体的漂白甘油。这两种产品使组织被存储的时间，在4℃下长期使用。 </li><li>捕捉利用共聚焦显微镜荧光每个人的图片。使用激发波长为荧光团和过滤器使用的表3中。 </li><li>使用根据用于捕捉图像共焦的类型适当的软件执行计算机辅助图像分析。 </li></ol>

<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. J., 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=Colonic+enteric+nervous+system+analysis+during+parenteral+nutrition.">Colonic enteric nervous system analysis during parenteral nutrition.</a> J Surg Res. 184 (1), 132-137 (2013).</li><li>Erickson, C. S., Zaitoun, I., Haberman, K. M., Gosain, A., Druckenbrod, N. R., Epstein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sacral+neural+crest-derived+cells+enter+the+aganglionic+colon+of+Ednrb(-/-)+mice+along+extrinsic+nerve+fibers.">Sacral neural crest-derived cells enter the aganglionic colon of Ednrb(-/-) mice along extrinsic nerve fibers.</a> J Comp Neurol. 20 (3), 620-632 (2011).</li><li>Zaitoun, I., Erickson, C. S., 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=Altered+neuronal+density+and+neurotransmitter+expression+in+the+ganglionated+region+of+Ednrb+null+mice:+implications+for+Hirschsprung's+disease.">Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.</a> Neurogastroenterol Motil. , (2013).</li><li>Margolis, K. G., Stevanovic, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neuronal+density+contributes+to+the+severity+of+intestinal+inflammation.">Enteric neuronal density contributes to the severity of intestinal inflammation.</a> Gastroenterology. 141 (2), 588-598 (2011).</li><li>Qu, Z. -. D., Thacker, M., Castelucci, P., Bagy&#225;nszki, M., Epstein, M. L., Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+analysis+of+neuron+types+in+the+mouse+small+intestine.">Immunohistochemical analysis of neuron types in the mouse small intestine.</a> Cell Tissue Res. 334 (2), 147-161 (2008).</li><li>Bian, X. -. C., Bornstein, J. C., Bertrand, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+transmission+at+functionally+distinct+synapses+in+descending+reflex+pathways+of+the+rat+colon.">Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon.</a> Neurogastroenterol Motil. 15 (2), 161-171 (2003).</li><li>Johnson, C. D., Epstein, M. 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>

The authors have nothing to disclose.

我们的实验室和其他人已经表明，在HSCR肠缺陷不限于无神经节结肠但近侧延伸，连成神经节小肠5,15,16。这些改变包括改变在ENS神经元密度和神经递质的表型，并且可以考虑动力障碍已被观察到的患者HSCR。我们利用上述技术，我们在努力了解ENS形成的决定因素。具体地，这些技术已被用来可视化神经元一氧化氮合成酶（nNOS的）神经元，血管活性肠肽神经元（VIP），以及胆碱乙酰转移酶（C...

一个发挥作用的肠神经系统（ENS），控制运动，营养吸收和局部血流量，是生活中必不可少的1。该ENS是由神经嵴细胞（NCC）的增殖，迁移和定居的肠道，在那里他们分化成神经节含有神经元和神经胶质细胞形成。先天性巨结肠症（文中HSCR，在线孟德尔遗传），一个multigeneic先天性疾病与1 4000活产儿发病，可以被认为是原型病研究打乱ENS形成。在HSCR，NCC无法迁移并定居于远端肠2的可变长度。此外，其他常见的胃肠道（GI）中的儿科人群发育缺陷，如肛门直肠畸形，肠atresias，和运动障碍是与在基本ENS功能紊乱有关，并有可能与微妙，低估，解剖变化和功能性变化有关在ENS 3-6。因此，技术，使我们能够理解的ENS形成发展的决定因素可能会阐明其发病机制及小儿胃肠道疾病的潜在治疗轻。 以下迁移和殖民化，NCC分化为神经元特异性神经递质的表型标记。胆碱能神经元包括肠神经元7的约60％，并且可以通过染色胆碱乙酰转移酶（ChAT的），合成酶的兴奋性神经递质乙酰胆碱来检测。在历史上，试图可视胆碱能神经元是由不同的针对中枢神经系统的抗体的抗原特异性混淆（CNS）的ChAT的与外周神经系统（PNS）的ChAT的8-10。然而，针对胎盘胆碱抗体识别中枢和外周胆碱11-13，和我们最近所描述的技术，允许visuaENS胆碱能神经元的高灵敏度补肾中药较早发展比已取得的ChAT记者线14。 在这里，我们提出了解剖，固定小鼠胚胎胃肠道和免疫可视化ENS神经递质表达的神经元的技术。在这些研究中，我们已经利用交配R26R聊天的Cre小鼠：floxSTOP：tdTomato动物生产聊天的Cre; R26R：floxSTOP：tdTomato小鼠（整个手稿定义 ​​为聊天的Cre tdTomato）。这些动物，然后用纯合子聊天GFP报告小鼠交配，获得表达的小鼠都来检测胆碱表达荧光14记者。这两个记者动物是在C57BL / 6J背景和市售（Jackson实验室，巴港，缅因州）。

<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.

我们先前描述了小鼠表达GFP和tdTomato荧光记者，检测ChAT的表达14的产生。简单地说， 聊天的Cre小鼠交配R26R：floxSTOP：tdTomato动物生产聊天的Cre; R26R：floxSTOP：tdTomato小鼠（称为聊天的Cre tdTomato）。这些动物处死后交配的纯合的ChAT-GFP报道小鼠。胚胎中分离和组织之前被固定进行解剖并如上述进行免疫染色，并在表1中列出的抗体。远端小肠（SI）?...

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.

免疫组化可视化小鼠肠神经系统的发展

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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11.3K 次观看.  University of Wisconsin School of Medicine and Public Health. 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.