Name: organ culture and whole mount immunofluorescence staining of mouse wolffian ducts
Uploaded: 2017-01-13T15:00:00
Description: Watch this Scientific Journal Video about Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts at JoVE.com

我们要感谢妇科肿瘤学组的成员对这个手稿的批判性阅读。这项工作部分由来自国家健康与医学研究理事会，澳大利亚研究理事会和癌症研究所新南威尔士州（PST）的资金支持。 MK是纽卡斯尔研究生研究奖学金的大学的收件人。

动物保健和实验过程是按照纽卡斯尔大学的动物护理和伦理委员会的指导方针进行，并确认新南威尔士州动物研究法，新南威尔士州动物研究的管理和照顾澳大利亚代码用动物用于科学目的的。对小鼠进行的所有的程序是由纽卡斯尔大学的动物护理和伦理委员会批准。 1.时间交配<ol><li>对6 - 8周龄雄性和雌性小鼠之前，为了日光周期结束。 </li><li>检查女性的阴道存在插头?...

<ol>
	<li>Joseph, A., Yao, H., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+morphogenesis+of+the+Wolffian/epididymal+duct,+more+twists+and+turns.">Development and morphogenesis of the Wolffian/epididymal duct, more twists and turns.</a> Dev Biol. 325, 6-14 (2009).</li><li>Murashima, A., Xu, B., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+normal+and+abnormal+development+of+the+Wolffian/epididymal+duct+by+using+transgenic+mice.">Understanding normal and abnormal development of the Wolffian/epididymal duct by using transgenic mice.</a> Asian J Androl. 17, 1-7 (2015).</li><li>Carroll, T. J., Park, J. S., Hayashi, S., Majumdar, A., McMahon, 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=Wnt9b+plays+a+central+role+in+the+regulation+of+mesenchymal+to+epithelial+transitions+underlying+organogenesis+of+the+mammalian+urogenital+system.">Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system.</a> Dev Cell. 9, 283-292 (2005).</li><li>Kumar, M., Syed, S. M., Taketo, M. M., Tanwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Wnt/&#946;catenin+signalling+is+essential+for+epididymal+coiling.">Epithelial Wnt/&#946;catenin signalling is essential for epididymal coiling.</a> Dev Biol. 412, 234-249 (2016).</li><li>Murashima, A., 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=Essential+roles+of+androgen+signaling+in+Wolffian+duct+stabilization+and+epididymal+cell+differentiation.">Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation.</a> Endocrinology. 152, 1640-1651 (2011).</li><li>Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+limitations+of+genetically+modified+mouse+models+in+drug+discovery+and+development.">Applications and limitations of genetically modified mouse models in drug discovery and development.</a> Curr Drug Metab. 9, 419-438 (2008).</li><li>Rak-Raszewska, A., Hauser, P. V., Vainio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+In+Vitro+Culture:+What+Have+We+Learned+about+Early+Kidney+Development?.">Organ In Vitro Culture: What Have We Learned about Early Kidney Development?.</a> Stem Cells Int. 959807, 1-16 (2015).</li><li>Hirashima, T., Adachi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedures+for+the+Quantification+of+Whole-Tissue+Immunofluorescence+Images+Obtained+at+Single-Cell+Resolution+during+Murine+Tubular+Organ+Development.">Procedures for the Quantification of Whole-Tissue Immunofluorescence Images Obtained at Single-Cell Resolution during Murine Tubular Organ Development.</a> PLoS One. 10, 0135343 (2015).</li><li>Chan, W. Y., Blomberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+Development:+Methods+and+Protocols.">Germline Development: Methods and Protocols.</a> Methods Mol Biol. 825, (2012).</li><li>Karner, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tankyrase+Is+Necessary+for+Canonical+Wnt+Signaling+During+Kidney+Development.">Tankyrase Is Necessary for Canonical Wnt Signaling During Kidney Development.</a> Dev Dyn. 239, 2014-2023 (2010).</li><li>Traykova-Brauch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+versatile+system+for+acute+and+chronic+modulation+of+renal+tubular+function+in+transgenic+mice.">An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.</a> Nat Med. 14, 979-984 (2008).</li></ol>

在器官培养系统具有比传统细胞培养系统的许多优点。这个系统保留不同的细胞类型之间的原始结构关系和相互作用。它模仿体内系统，并提供比细胞培养的研究，其中利基因素是缺乏更准确的信息。使用器官培养系统，甚至有过用整个动物的优势。这些措施包括使用整个动物，易于打理和器官文化系统的维护等 。而且成本较高，各种药物制剂或药物可以孤立/培养器官和器官的直?...

男性生殖系统主要包括睾丸，站点生殖细胞的发育和分化，并且是所必需的成熟，运输和储存精子的一个复杂的管道系统。附睾是连接与输精管睾丸及主要参与生殖细胞1，睾丸后的发育和成熟的管状器官。高度盘绕成年小鼠附睾从一个简单而直接前体管，沃尔夫管（WD）1发展。精子采集施肥雌性生殖细胞2的能力附睾的复杂性和绕制线圈的结构是至关重要的。对于男性生育这样的重要器官如何开发和进入形状还不是很清楚。多种信号途径已经显示在WD的发展涉及如Wnt信号通路3,4和雄激素信号传导途径5。我们最近的工作已经建立BAL的要求高级操作的Wnt胎儿发育期间4信令WD卷取。然而，需要进一步的研究来理解附睾上皮内和与间质细胞参与出生后的附睾卷取，细胞分化和细胞 - 细胞通信的机制。 转基因小鼠模型已经被证明是对的底层各器官系统6的发育和疾病的分子机制的识别/验证的有力工具。尽管它的广泛使用，存在转基因小鼠模型的显著的局限性，包括靶向小鼠突变体的不可预测的表型与问候推定基因功能，而在无效突变体在一些模型中没有表型，遗传和环境因素的影响，劳动密集和耗时开发小鼠模型的过程。因此，只有从基因改性的研究结果的意义的解释田间小鼠模型并不总是直截了当6。这些限制可通过使用体外器官培养系统，该系统使我们能够灵活操纵在受控的培养条件在实时多个信号通路来克服。器官培养体系是理解整个器官7的生理和病理作用。此外，与荧光标记的抗体染色的整个器官的成像，使我们能够可视化感兴趣的标志物的三维情况下，因为它存在于生物体内，从而提供更好的理解，该机关的形状，结构，和功能8。 这里，我们已经描述了用于小鼠胚胎生殖腺嵴的隔离， 在体外培养和WDS的整装免疫荧光成像的方法中，可应用来回答各种各样的关于管状器官如WDS的形态发生的问题。在这个协议中，我们从15.5天孤立的小鼠胚胎泌尿生殖脊交配后（DPC）怀孕水坝和培养他们3天之后免疫染色的上皮细胞标记物（细胞角蛋白8，CK8），细胞增殖标志物（磷酸化组蛋白3，PH3）和有源βcatenin。

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">24 well plates</td>
			<td style="width:195px;">Corning, USA</td>
			<td style="width:136px;">3524</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td style="width:195px;">GE Healthcare Life Sciences, USA</td>
			<td style="width:136px;">SH30031.02</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">D8437</td>
			<td style="width:191px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:195px;">Interpath,&#160; Australia</td>
			<td style="width:136px;">SH30034.02</td>
			<td style="width:191px;">10% FBS used in culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">L-Glutamine</td>
			<td style="width:195px;">GE Healthcare Life Sciences, USA</td>
			<td style="width:136px;">SH30034.01</td>
			<td style="width:191px;">1% concentration used</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:247px;">Penicillin-Streptomycin</td>
			<td style="width:195px;">Gibco, USA</td>
			<td style="width:136px;">15070-063</td>
			<td style="width:191px;">1% concentration used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Whatman Nuclepore Polycarbonate Track-Etch&#160;</td>
			<td style="width:195px;">Whatman, USA</td>
			<td style="width:136px;">110409</td>
			<td style="width:191px;">Membrane (0.8 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">IWR1</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">10161-5mg</td>
			<td style="width:191px;">100 &#181;M concentration used</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Paraformaldehyde (PFA)</td>
			<td style="width:195px;">Electron Microscopy Sciences, USA</td>
			<td style="width:136px;">15710</td>
			<td style="width:191px;">16% stock, 4% in PBS for use. TOXIC - wear gloves and cannot 
			be disposed of in the sink</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Ethanol</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">214-20L PL</td>
			<td style="width:191px;">Absolute, make 25, 50 and 75% with milliQ water.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Tween-20</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">2509-500ml</td>
			<td style="width:191px;">Very viscous, careful while dispensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Triton X-100</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">T8787-50ml</td>
			<td style="width:191px;">Very viscous, careful while dispensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Di-Sodium Hydrogen Phosphate</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">621-500mg</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Sodium Di-hydrogen Phosphate</td>
			<td style="width:195px;">Ajax Finechem, USA</td>
			<td style="width:136px;">4745-500g</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Sodium Chloride</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">465-500g</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:247px;">Cytokeratin8/Troma I</td>
			<td style="width:195px;">Developmental Studies Hybridoma Bank, USA (DHSB)</td>
			<td style="width:136px;">TROMA-I</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">active &#946;catenin&#160;</td>
			<td style="width:195px;">Cell Signalling Technology,&#160; USA</td>
			<td style="width:136px;">D13A1</td>
			<td style="width:191px;">1:200 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:195px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Phospho-Histone3</td>
			<td style="width:195px;">Millipore, MA, USA</td>
			<td style="width:136px;">06-570</td>
			<td style="width:191px;">1:200 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 Goat anti-rabbit IgG</td>
			<td style="width:195px;">Jackson ImmunoResearchLabs,&#160; USA</td>
			<td style="width:136px;">111-545-047</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:247px;">Alexa594 Goat anti-Rat IgG</td>
			<td style="width:195px;">Jackson ImmunoResearchLabs,&#160; USA</td>
			<td style="width:136px;">112-585-072</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Glycerol</td>
			<td style="width:195px;">Thermo Fisher Scientific, USA</td>
			<td style="width:136px;">242-500ml</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">n-Propyl galate</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">2370</td>
			<td>-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI)</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">D9564-10mg</td>
			<td style="width:191px;">Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:195px;">Thermo Fisher Scientific, USA</td>
			<td style="width:136px;">2225-500ml</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cover Slips (24x50 mm)</td>
			<td style="width:195px;">Lomb Scientific</td>
			<td style="width:136px;">CS24501GP</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

organ culture and whole mount immunofluorescence staining of mouse wolffian ducts

输卵管形态是大多数哺乳动物的器官，包括男性生殖系统的发展的基本要求。附睾，男性生殖道的一个组成部分，是负责精子储存，成熟和运输。成人附睾是一个高度盘管，从被称为沃尔夫管（WD）一个简单而直接的胚胎前体的发展。附睾的正确卷取对男性生育能力至关重要，因为精子在睾丸未能受精卵母细胞。然而，负责附睾发展和卷取的机制尚不清楚，部分原因是由于缺乏整个器官培养和成像方法。在这项研究中，我们描述了在体外培养系统和整装免疫协议，以更好地可视化的WD卷取和发展，这也可应用于研究其他管状器官的过程。

在开发过程中，对WD经历，其中一个简单的直管被转换成一个高度复杂和盘绕管显著变化。使用上述方法，WDS接受在文化条件类似的转变。在这里，我们已经表明，从3天（ 图3）培养的WDS的结果。解剖涉及WD形态的分子机制，培养基可以补充有抑制剂和活化剂靶向不同的信号通路。 图3C示出了Wnt信号的存在后培养3天开卷WD信令特异性抑制剂，IWR1 10。箭头标示为卷?...

Watch this Scientific Journal Video about Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts at JoVE.com

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

我们目前的鼠标沃尔夫管（WD）的分离，培养的方法。我们也表现出与荧光标记的抗体培养/新鲜分离的WDS的整装免疫的详细过程。总之，这些技术使WD的发展，卷取和分化的研究。

器官培养和鼠标沃尔弗导管的全山免疫染色

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This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

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小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

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Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...