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凌晨（或8.00之前AM），每一天。塞日被认为是0.5 DPC。 </li><li>转移与阴道塞女到一个新的笼子（无男）和标签塞的日期。 </li></ol> 2.小​​鼠胚胎生殖腺嵴的分离<ol><li>分离小鼠胎如下所述在9从15.5 DPC怀孕女性集成电路生殖腺嵴如所述的，具有一些修改。 </li><li>作为中午的时候怀孕女性被认为是进入到了下午怀孕的第二天，之前进行清扫。 </li><li>用牺牲颈椎脱位15.5 DPC怀孕女性（或按机构动物伦理委员会批准的协议）。 </li><li>保留在其上的吸收性薄纸背面的鼠标和喷雾用70％乙醇腹部的腹部侧。 </li><li>提起下腹部皮肤（腹侧）用钳子和使用手术剪作外侧切口，泌尿生殖开放的结束肋骨延伸。拉扯皮肤朝着动物的头以露出腹部。 </li><li>切开腹部肌肉手术剪，露出腹腔。抓住只用宫颈钳以上妊娠子宫并进行剪切。现在，通过举办对T解除子宫Ó膀胱和切开子宫阔韧带随后在子宫输卵管接合部削减从腹膜附件分离子宫。 </li><li>转移子宫到含有冰冷的HBSS（Hank氏平衡盐溶液）的50mL管中。用冰冷的HBSS轻轻冲洗妊娠子宫去除多余的血。 </li><li>保留子宫在含有冰冷的HBSS培养皿，并保持在冰上。切开子宫壁，取出胚胎。保持胚胎在一个新的培养皿HBSS冰上。 </li><li>取一干净无菌纸巾，把它放在一个新的培养皿。喷在棉纸70％的乙醇。保持上侧面本棉纸胚胎，并使用在方向无菌刀片如图1中所示的虚线从下腹切开它。 </li><li>由钉住脊柱固定在无菌海绵基地的雏形。 </li><li>将它放置在解剖体视显微镜下，并沿腹中线经过精心切割。取出肝脏和肠道。鼠标泌尿生殖系统（肾，输尿管，睾丸，WD，和输精管）现在应该是可见的。 </li><li>剪下睾丸和WD，清新的培养皿保持与HBSS，冰上。取出的WD和睾丸，切接近其附着到尿道输精管和切断从引带的WD的下部的附件。 </li><li>池睾丸和WDS从同一组的胚胎（这可能根据实验计划变化）。 </li></ol> 3.胚胎性腺岭南文化（图2） <ol><li>通过用10％FBS（胎牛血清），1％青霉素/链霉素和1％L-谷氨酰胺补充的DMEM / F12中培养胚胎生殖腺嵴制备介质。温热介质至37℃的水浴中。 </li><li>每孔加入24孔培养板的媒体300微升。 </li><li>取新鲜培养皿，把无菌HBSS一小滴就可以了。把聚碳酸酯轨道蚀刻膜（0.881;在此下降的HBSS其光亮的表面所面临的HBSS米）。 </li><li>使用清洁的镊子把两个WDS和性腺在膜（在其表面粗糙，朝上），并使用无菌湿巾/吸收纸除去HBSS的最大量。请勿用吸水纸组织;否则，会损坏组织。 </li><li>确保无论是WDS也不性腺互相接触。否则，他们将随后的孵化过程中粘在一起。 </li><li>在含有300微升培养基，并培养他们在空气 - 介质界面24孔板的孔转移与组织中的膜。在WDS的囊性生长膜结果太多的培养基。 </li><li>孵育所述板在37℃培养箱，在5％CO 2的存在。 </li><li>每天更换培养基。要改变用吸管介质，请从井第一介质，然后添加预热的新鲜培养基的300微升。 注意:为了研究关于WD形态不同信号通路的作用，在所要求的浓度的化学活化剂和/或抑制剂可以添加到培养基中。 </li><li>文化组织3天。在3天，从15.5 DPC胚胎收集解开WDS变成非常费解管（ 图3B）。在加入的Wnt抑制剂，IWR1，的结果以抑制WD卷取（ 图3C）的。 </li><li>收获这些组织采取培养皿用冰冷的PBS中。以培养性腺和WD膜转移到培养皿。该膜将浮在PBS中。按膜击沉它在PBS，并采取了性腺和WD。 </li><li>用4％多聚甲醛（PFA）O / N的组织在4℃或在RT 1小时。如果需要，采取WDS的亮场图像固定前。 </li></ol> 4.整装免疫<ol><li>洗固定组织3倍用PBS-T（PBS + 1％的Triton X-100）与缓慢摇动，每次10分钟，在室温。 </li><li>脱水组织在梯度乙醇（25％，50％，75％和100％），每次10分钟，在4℃，用缓慢摇动（〜30转）。在该步骤中，组织可以被存储在4℃下在75％的乙醇。 <ol><li>要更改乙醇，保持不受干扰2分钟的组织，让组织中安顿下来。取出尽可能多的上清液尽可能然后添加乙醇的下一个更高的浓度。为避免组织造成损害的，枪头不应在任何阶段触摸组织。 </li></ol></li><li>以下脱水，再水合组织在梯度乙醇（100％，75％，50％和25％），每次10分钟，在4℃，用缓慢摇动。 </li><li>洗用PBS-T（PBS + 0.1％的Triton X-100），4倍，每次20分钟，在室温的组织中，并轻轻摇动（〜30转）。 </li><li>用封闭缓冲液阻断在RT 1小时的组织（PBS + 1％BSA + 0.2％脱脂奶粉+ 0.3％的Triton X-100）。 </li><li> AFTEř阻断，转移组织至初级抗体溶液（在封闭缓冲液中稀释）中，用温和的摇摆（〜30rpm下）孵育O / N在4℃。使用的初级抗体的稀释液在材料表中描述。 </li><li>第二天，洗涤组织用PBS-MT（PBS + 2％无脂干奶粉+ 0.5％吐温-20），4倍，每次30分钟，在RT，用温和的摇摆（〜30转）。 </li><li> 250（此步骤需要对各个抗体优化）在封闭缓冲液中，并在室温下孵育1小时，用温和的摇摆:以下洗涤工序中，以1:1的稀释度加入二级抗体。 </li><li>从现在开始保持与铝箔覆盖，以防止暴露在光线继发抗体用对光敏感荧光团组织。 </li><li>洗组织3倍用PBS-T（PBS + 0.1％吐温-20），每次30分钟，用温和的摇摆。 </li><li>准备幻灯片安装染色组织。 <ol><li>对于这一点，切玻璃盖玻片成用金刚石笔和细条坚持两条了另一种使用指甲油搏命。让两个边界和地方组织在这两个边界之间。 </li><li>添加含有DAPI安装介质放在盖玻片。在盖玻片的角落应用指甲油，将其固定的幻灯片。 注:组织现在已经准备好进行成像。存储这些幻灯片在-20℃。 </li></ol></li></ol>

<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>

作者什么都没有透露。

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

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

organ-culture-whole-mount-immunofluorescence-staining-mouse-wolffian

Research

JoVE Journal

Developmental Biology

10.7K 次观看.  University of Newcastle. 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。 

视频: Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

科研

发育生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

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

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

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 ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

小鼠肝外胆管有机体的产生

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