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接反应，而不是整个身体可以进行测试研究。 有迹象表明，需要考虑胚胎WDS的培养成功的几个关键步骤。组织必须精心收集而不伤害它们的完整性，受损的组织不生长良好，成为囊状。的介质上的顶部过量过滤器也导致WDS的囊性增长。我们进行组织分离培养的组织培养罩外，没有任何污染。培养基的制备和加入到组织培养罩内部24孔板。因此，采用适当的技术，快速的工作，并采取必要的防范措施，组织培养罩外收集WDS可以是没有任何污染的培养。而收获培养组织，应小心，因为它们是非常脆弱的，不应该与钳子直接抓住。组织应该代替在PBS中的薄膜是钳子的两个臂之间形成被拾起。 我们还在这个手稿中描述了详细的整装免疫荧光法。使用共聚焦显微镜或立体整装染色后组织Z-叠加成像提供了一个优势，通过部分不连续切片和染色多个幻灯片整个组织。因此，它提供更好的主意靶蛋白的表达的位置。抗体渗透是在具有高纤维/细胞外基质含量组织导致较低信号困难。再通透性和一抗孵育时间的增加可能是在这种情况下非常有用。整装染色的另一缺点是，在单细胞水平分辨率是很难达到的，并可能需要专门的目标/显微镜。我们在4％PFA进行两个1小时，O / N固定，并发现，这些方法对于大多数在我们的实验室，包括CK8，PH 3，和活性βcatenin抗体测试的抗体的效果很好。我们还成功地与共定位PH3或主动βcateninCK8。对于共定位，同时加入两种标记的初级抗体。这些抗体必须在不同物种得到提升。在整个安装免疫荧光法，悉心照顾应该添加或删除时，解决方案，组织可以easil采取Ÿ绘制成枪头/移液管和卡住的塑料。为了避免这种情况，一个立体镜或解剖显微镜下更改与200μL的枪头溶液，并在管的底部离开的溶液一点点。我们建议在干净透明的玻璃烧杯收集垃圾的解决方案。每种溶液的变化后，计数组织在一个立体镜下管的数目。在组织中的意外损失的情况下，他们可以从该玻璃烧杯中回收。用于传输解决方案不应该触碰的组织，因为这会破坏组织，让他们不适合用于成像枪头。 对于安装彩色WDS，我们采用腔幻灯片那样直接把盖玻片上WDS的顶部可以压缩并破坏它们的形态。取决于组织的厚度，空腔的深度可以容易地通过改变所用盖玻片条的数目操纵。滑动preparatio的这种方法n是经济，因为它只需要盖玻片和金刚石笔。成像也可以盖片，因为它是易于定向和移动染色WDS它们下盖玻片固定之前做过。随着整个装载免疫，Z-叠加成像提供了更好的图像，并提供更多的信息。来自不同处理组拍摄图像，曝光应保持不变。

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><tbody><tr><td>24-well plates</td><td>Corning, USA</td><td>3524</td><td>can be purchased from other vendors</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>GE Healthcare Life Sciences, USA</td><td>SH30031.02</td><td>can be purchased from other vendors</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td><td>Sigma, USA</td><td>D8437</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Interpath,&amp;nbsp; Australia</td><td>SH30034.02</td><td>10% FBS used in culture medium</td></tr><tr><td>L-Glutamine</td><td>GE Healthcare Life Sciences, USA</td><td>SH30034.01</td><td>1% concentration used</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco, USA</td><td>15070-063</td><td>1% concentration used</td></tr><tr><td>Whatman Nuclepore Polycarbonate Track-Etch&amp;nbsp;</td><td>Whatman, USA</td><td>110409</td><td>Membrane (0.8 &amp;micro;m)</td></tr><tr><td>IWR1</td><td>Sigma, USA</td><td>10161-5mg</td><td>100 &amp;micro;M concentration used</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences, USA</td><td>15710</td><td>16% stock, 4% in PBS for use. TOXIC - wear gloves and cannot&lt;br/&gt; be disposed of in the sink</td></tr><tr><td>Ethanol</td><td>Thermo Scientific Fisher, USA</td><td>214-20L PL</td><td>Absolute, make 25, 50 and 75% with milliQ water.</td></tr><tr><td>Tween-20</td><td>Thermo Scientific Fisher, USA</td><td>2509-500ml</td><td>Very viscous, careful while dispensing</td></tr><tr><td>Triton X-100</td><td>Sigma, USA</td><td>T8787-50ml</td><td>Very viscous, careful while dispensing</td></tr><tr><td>Di-Sodium Hydrogen Phosphate</td><td>Thermo Scientific Fisher, USA</td><td>621-500mg</td><td>can be purchased from other vendors</td></tr><tr><td>Sodium Di-hydrogen Phosphate</td><td>Ajax Finechem, USA</td><td>4745-500g</td><td>can be purchased from other vendors</td></tr><tr><td>Sodium Chloride</td><td>Thermo Scientific Fisher, USA</td><td>465-500g</td><td>can be purchased from other vendors</td></tr><tr><td>Cytokeratin8/Troma I</td><td>Developmental Studies Hybridoma Bank, USA (DHSB)</td><td>TROMA-I</td><td>1:250 in blocking buffer</td></tr><tr><td>active &amp;beta;catenin&amp;nbsp;</td><td>Cell Signalling Technology,&amp;nbsp; USA</td><td>D13A1</td><td>1:200 in blocking buffer</td></tr><tr><td>phospho-Histone 3</td><td>Millipore, MA, USA</td><td>06-570</td><td>1:200 in blocking buffer</td></tr><tr><td>Alexa488 Goat anti-rabbit IgG</td><td>Jackson ImmunoResearchLabs,&amp;nbsp; USA</td><td>111-545-047</td><td>1:250 in blocking buffer</td></tr><tr><td>Alexa594 Goat anti-Rat IgG</td><td>Jackson ImmunoResearchLabs,&amp;nbsp; USA</td><td>112-585-072</td><td>1:250 in blocking buffer</td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific, USA</td><td>242-500ml</td><td>can be purchased from other vendors</td></tr><tr><td>n-Propyl galate</td><td>Sigma, USA</td><td>2370</td><td>-</td></tr><tr><td>2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI)</td><td>Sigma, USA</td><td>D9564-10mg</td><td>Use to stain nucleic acids (DNA)</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Thermo Fisher Scientific, USA</td><td>2225-500ml</td><td>Solvent</td></tr><tr><td>Cover Slips (24 x 50 mm)</td><td>Lomb Scientific</td><td>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。箭头标示为卷曲（ 图3B）和解开WDS（ 图3A和C）指示的Wnt信号导致抑制WD卷取（ 图3C）的抑制。因此，该培养系统可以用来解剖DEVEL中所涉及的分子机制WDS的opment（或其他器官）。 要标记不同的细胞类型，我们进行了对WDS整装免疫染色（ 图4）。在这里，我们目前的数据验证了采用该协议标签骨架，细胞质和核蛋白质。 图4A表示细胞角蛋白8（CK8，上皮细胞的标记物）WDS的免疫染色（由箭头标记）培养3天。我们还应用了相同的协议通过免疫染色PH3（磷组蛋白3，细胞增殖标记物），以评估细胞增殖。 图 4B示出的PH3整装免疫染色的代表性图像。箭头标记为绿色圆点代表PH3正因此，增殖细胞。 图4C示出的免疫染色，用于对从18.5 DPC小鼠胚胎新鲜分离WDS活性β连环蛋白。在图4D的阴性对照（IgG对照）显示没有染色。这些成果突出这个整装免疫染色的协议，这也可用于许多不同的抗体的稳定性。 <img alt="图1" src="/files/ftp_upload/55134/55134fig1.jpg" /> 图1图解切口部位为胚胎性腺的分离的从15.5 DPC胚胎。白色虚线标记用于使切口在15.5 DPC胚胎分离泌尿生殖脊的部位。 <a href="http://ecsource.jove.com/files/ftp_upload/55134/55134fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55134/55134fig2.jpg" /> 图2.流程图描述用于器官培养逐步过程 。 <a href="http://ecsource.jove.com/files/ftp_upload/55134/55134fig2large.jpg" target="_blank">请点击此处为vi</a>EW这个数字的放大版本。 <img alt="图3" src="/files/ftp_upload/55134/55134fig3.jpg" /> 图3.在培养条件下正常WD形态。 （A）睾丸和WD从15.5 DPC胚胎中分离。 （ 二 ）卷材WD后DMSO（对照）的存在3天的文化。与IWR1治疗（一Wnt信号抑制剂）观察（C）无WD卷取。箭头标记WD; T，标志着睾丸。杆等于100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55134/55134fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55134/55134fig4.jpg" /> 图4.整装WDS的免疫标记。 （A - B）WDS伊索拉的整装免疫泰德从15.5 DPC胚胎和3天的CK8（A）和PH3（B）培养。 （ 三 ）积极βcatenin对WD整装免疫18.5 DPC胚胎中分离。 （D）阴性对照的18.5 DPC WD（IgG对照）主动βcatenin显示没有染色。盘绕箭头WD标志; T，标志着睾丸。杆等于100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55134/55134fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

科研

发育生物学

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

癌症研究

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

<meta charset="utf8"></head><body>类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

类器官的三维成像以研究 离体 器官发生过程中的原发性纤毛发生

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

遗传学

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, ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

下游免疫污染分析小鼠水母上皮周期特定阶段的转化-辅助解剖

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

神经科学

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

生物学

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

器官培养和鼠标沃尔弗导管的全山免疫染色

摘要

探索更多视频

运用

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

基于细胞基和细胞外矩阵的Murine睾丸器官生成

下游免疫污染分析小鼠水母上皮周期特定阶段的转化-辅助解剖

全山解剖和成年小鼠耳蜗的免疫

老鼠胎儿整肠文化系统

对小鼠乳腺冰冻切片间接免疫荧光

器官培养和鼠标沃尔弗导管的全山免疫染色

摘要

探索更多视频

运用

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

类器官的三维成像以研究 离体 器官发生过程中的原发性纤毛发生

类器官的三维成像以研究 离体 器官发生过程中的原发性纤毛发生

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

基于细胞基和细胞外矩阵的Murine睾丸器官生成

下游免疫污染分析小鼠水母上皮周期特定阶段的转化-辅助解剖

全山解剖和成年小鼠耳蜗的免疫

老鼠胎儿整肠文化系统

对小鼠乳腺冰冻切片间接免疫荧光

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生