We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

注:所有小鼠，采用经美国密歇根医学院单位的大学实验动物医学根据大学委员会关于利用动物和护理的指导方针的协议处理人道。 1，全器官培养系统<ol><li>传媒与文化板块的准备<ol><li>在组织培养罩，从股市瓶取出5毫升BGJb媒体加入5 ml青霉素/链霉素的。准备媒体工作的股票在50毫升离心管中，加入1毫升5毫克/毫升抗坏血酸49毫升BGJb媒体（含青霉素/链霉素）。 </li><li>加入1 ml下面各转孔的准备了6孔培养板。 注意:如果不使用所有的6转孔，将多余的转孔进入一个新的无菌6孔培养板，供以后使用。再加上工作BGJb媒体每三个10厘米无菌培养皿中3毫升。 </li></ol></li><li>解剖准备<ol><li>浸泡解剖工具，70％ETHA北环线，一定要保持清洁工具在工作时。 </li><li>设置了一个大冰桶的解剖区域，并放置在6孔培养板和10毫米培养皿中与BGJb介质上的冰。冰一样多，而解剖和准备肠道对文化工作成为可能。 </li><li>麻醉的成年雌性小鼠与异氟烷（100微升）之前安乐死颈椎脱位胎儿肠道的收获室。 </li><li>之后，从他们的母亲收获的胎儿，舞台仔细按照Theiler分期图表13每胎。不要依赖于天后性交，以确定每胎为最多24小时的发育阶段变化的时代中的一个垃圾例行观察。 </li></ol></li><li>胎儿肠道的解剖<ol><li>之前去除肠子安乐死胎儿被斩首。 </li><li>解剖在1×Dulbecco氏磷酸盐缓冲盐水（DPBS）各肠然后PLACE成10cm的培养皿与工作BGJb媒体。 </li><li>小心地从胃健脾和胃和十二指肠上部的胰腺。 </li><li>轻轻分开网膜照顾到离开附尽可能和分离的连接仅足够大网膜，以允许在肠道被拉直。 注:对于比E14.5老年人或那些被培养超过24小时，肠，轻轻分开肠道分成3个相等的段，以允许腔内容物的流动通过肠和从切割端排出。然而，对于培养开始之前，E13.5，等待培养直到24小时后分离出肠的3个区段，以使浆膜到正常生长在肠的长度之前。 </li><li>用吸管口在肠道件转移到培养皿（1.1.2）的转孔。 </li><li>根据需要，使它们径直穿过transwel轻轻地重新定位肠升。 注意:一旦肠道开始通过管道移动管腔内容，在肠道内的任何扭结会导致备份和损害肠道。 </li></ol></li><li>文化与治疗<ol><li>从Transwell小下取出介质，加入700μl处理介质（工作介质添加了重组蛋白或药物抑制剂）的transwell小下。 </li><li>加入300μl的处理介质滴加于肠内的顶部，并允许它浸泡通过transwell小。重新定位的肠子是必要的。 </li><li>至少每日一次，或更频繁地更换处理介质取决于治疗试剂的半衰期。 </li></ol></li><li>治疗试剂的局部源制备蛋白浸泡琼脂糖珠<ol><li>悬浮琼脂糖珠在自己的存储容器，并转移100微升的琼脂糖珠到1.5 ml离心管中。 </li><li>填充管的其余容积用无菌1X博士osphate缓冲盐水（PBS）中并混合的珠子洗涤。将离心管中的齿条几分钟以使珠沉降至底部，然后从上珠粒顶端吸取掉PBS中。重新填充离心管中，用无菌1×PBS中，并重复洗涤过程两次以上。 </li><li>制备所关注的蛋白的两倍所需的治疗浓度。 </li><li>混合5微升的洗涤过的琼脂糖珠与5微升的2个蛋白存量，并轻轻搅拌中的蛋白质的珠。放置在冰上的管中1小时后，混合轻轻地每10-15分钟。 </li><li>把珠子上的肠子前在无菌1X PBS冲洗短暂的珠子。 </li></ol></li><li>琼脂糖珠安置<ol><li>轻轻将琼脂糖珠上使用吸管口用毛细管拉针肠子上。将珠子非常谨慎的粗糙处理会损害肠道，防止绒毛发展中的受伤部位。 </li><li>将两倍多珠需要进行分析，因为肠子会发生蠕动和大约一半的放置会滚下肠子在培养时间的珠子。 </li></ol></li><li>培养肠道内固定<ol><li>小心地从transwell小下面取出处理介质，并允许肠内空气干燥3分钟。 </li><li>加入1 ml固定液的transwell小室下2分钟，然后盖与2ml固定液为的固定时间剩余肠道的顶部。修正了4％多聚甲醛在o / n 4℃或2小时在室温。 </li></ol></li></ol> 2，修正了影像的肠子<ol><li> Wholemount成像<ol><li>将整个肠子（与内源性荧光）上用100微升1X DPBS的幻灯片。使用镊子轻轻地安排肠子。 </li><li>使用实验室的组织，以小心地取出DPBS，立即加入100微升70％的甘油，使组织MO再透明，提高成像的可能深度。 注:不是安装在70％甘油中的肠如果肠的进一步渗透是期望的，浸泡在几滴焦点清除溶液中的肠10-30分钟。吸管关闭对焦清晰的解决方案，并安装与安装清除肠道。 </li><li>每个四角盖玻片动用橡皮泥，拿起少量粘土使用的幻灯片和盖玻片之间的间隔，以保持盖玻片从压扁的肠道。 </li><li>密封盖玻片上施加，用棉签融化VALAP的幻灯片。 </li><li>图片上无论是直立或倒置的共聚焦显微镜的肠子。请参阅更详细的讨论。 </li></ol></li><li>固定肠子Vibratome切片（肠结构的剖视图） <ol><li>在7％琼脂糖嵌入肠子在小塑料模具（10×10×15毫米），并允许将琼脂糖块冷却和固化。 </li><li>从塑料模具和琼脂糖胶块与瞬间胶的vibratome收集阶段取出琼脂糖块。 </li><li>填用冰冷的1X DPBS收集阶段。 </li><li>切段在100-150微米之间的厚度。对于较厚的部分使用结算解决方案（在2.1.2节中描述），以可视化更深的横截面。 </li><li>倾从收集阶段vibratome节到6孔板中存储的阱，在4℃。 </li></ol></li><li>固定，vibratome切片组织免疫荧光染色<ol><li>将每口井5-12 vibratome节到24孔板与1X DPBS。 </li><li>删除1X DPBS和每孔加入500μl的通透的解决方案。轻轻摇动样品在室温下25分钟。 </li><li>透化后，用1×PBS洗涤样品3次。 </li><li>阻断样品中加入500μl封闭液中的至少30分钟。 </li><li>后堵，E交换网板用500μl稀释在封闭溶液中的初级抗体的阻断溶液并保持样品在4℃CO / N。 注:在冰冻和石蜡切片工作，以及一抗稀释液一般在vibratome部分工作得很好，但抗体需要核抗原修复一般不与本协议（ 例如，BrdU的）工作。 </li><li>翌日，每次用封闭溶液洗涤样品3次，持续15分钟。 </li><li>洗涤后，将500μl的稀释在封闭溶液中的二级抗体。包裹24孔板中的铝箔，以保护荧光团从光，并摇动样品45分钟至2小时，在室温。 </li><li>用1×PBS洗涤样品3次，每次15分钟，并在第2.4节中所述安装在滑轨的vibratome部分。 </li></ol></li><li>安装vibratome节<ol><li>准备幻灯片vibratome安装切割薄片双STICK胶带，并把它们沿着滑动的长边。 </li><li>小心地放置在幻灯片上的双面胶的5-10 vibratome节下降100μL1X PBS中。 </li><li>展开所有部分，并蔓延出来到对面的幻灯片单层。 </li><li>删除多余的琼脂糖或不均匀位，防止盖玻片从坐在平坦。 </li><li>小心使用实验室的组织来吸收多余的1X PBS来自各地的vibratome部分。 </li><li>加一滴含水安装介质上的vibratome部分的顶部，然后盖玻片。 </li><li>密封盖玻片上融化的VALAP应用，用棉签幻灯片。 </li><li>图片上无论是直立或倒置的共聚焦显微镜的vibratome部分。请参阅有关选择的成像系统的进一步的细节进行讨论。 </li></ol></li></ol> 3，现场全肠道的影像学肠子从fetuse收获ŠE12.5后，与所连接的大网膜原封不动，成功地利用上述系统开发绒毛文化。因此，本体外系统允许显影肠可重构的形态随时间的三维视图的活的z截面图像的捕捉。 <ol><li>建立实时成像<ol><li>使用瞬间胶，坚持个人的聚碳酸酯膜细网筛。这提供了一个支架支撑，以保持肠道到位直立共聚焦显微镜的镜片浸泡在下面。请参考讨论部分对选择实时成像显微镜进一步的细节。 </li><li>将网筛在中心以及文化板块。 </li><li>将解剖肠到聚碳酸酯膜，并在两端加一滴瞬间胶。仅使用足够的胶水贴上肠道，而不是大衣呢！ </li><li>添加酚红培养基免费聚碳酸酯以下膜在中心孔的培养板。 </li><li>加水至培养板的外边缘，以提供湿度。 </li><li>由于胎儿肠管发生在文化收缩蠕动，像波浪一样，加入100μl的1:在1X PBS溶液5甲苯噻嗪〜3毫升的媒体控制肠道而成像的运动。这个剂量控制肠道运动约8-10小时，不影响绒毛的发展。 </li><li>对于肠道的横向视图，嵌入在3-4％琼脂糖溶液活肠切成厚的部分（150-500微米）上vibratome。匹配与肠的刚度琼脂糖的刚性被切割和经验确定的发育​​阶段。一般情况下，3％的琼脂糖溶液可以很好地用于肠道年龄小于E14.5和4％的溶液可以很好地用于肠E15-E16。 </li><li>胶水vibratome部分以聚碳酸酯膜贴在网筛（如第3.1.1节），并培养第3.1.2-3.1.6所述。 </li><li>利用双光子共聚焦荧光显微镜（690-1,040纳米之间的激光激发）上直立架与可调表进行实时成像。 </li><li>双光子激光调谐到900nm的为用户提供了GFP信号的最佳分辨率深层渗透。它减少了损伤的组织，而成像。此外，激光的功率设定为尽可能低的排放量减少的组织损伤。通常，得到的GFP的良好激发，用12％的功率上的双光子激光。 </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

动态性和发展肠道复杂的组织相互作用的，需要三维可视化有这些形态发生事件的全面理解。随着不断变化的影像技术，能够检查绒毛形态进行了详细的发展/改善有了它，空间通信和交互的器官形成期的理解大大提高。 另一种方法培养整个肠子也进行了测试，但Transwell小系统仍是最可靠的更长的培养时间，并维持正常的集群和绒毛图案。三维培养系统（基质胶或胶原蛋白）?...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1308">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fine dissecting forceps&#160;</td>
			<td style="width:223px;">Fine Science Tools</td>
			<td style="width:133px;">&#160;11254-20</td>
			<td style="width:444px;">2 pairs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x sterile Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Gibco&#160;</td>
			<td>14040-133</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plates</td>
			<td>Costar&#160;</td>
			<td>3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 x 15 mm petri dishes</td>
			<td>Falcon</td>
			<td>&#160;451007</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td>
			<td>Corning Costar&#160;</td>
			<td>3428</td>
			<td style="width:444px;">6 inserts per plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BGJb media</td>
			<td>Invitrogen&#160;</td>
			<td>12591-038</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td>
			<td>Gibco</td>
			<td>&#160;15140</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic Acid</td>
			<td>Sigma&#160;</td>
			<td>A0278</td>
			<td style="width:444px;">make 5 mg/ml stock, filter, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td>
			<td>Fisher</td>
			<td style="width:133px;">&#160;21-180-10</td>
			<td style="width:444px;">remove the aspirator assembly and replace it with a 1000 &#181;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 inch glass pasteur pipets</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose beads</td>
			<td>BioRad&#160;</td>
			<td>153-7301</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary Tubes</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW100F-4</td>
			<td style="width:444px;">pull to needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td style="width:444px;">made in 1 x PBS, pH to 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live Imaging Materials</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture plates</td>
			<td>Falcon&#160;</td>
			<td>353037</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine mesh stainless steel screen</td>
			<td>purchase at hardware store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polycarbonate membranes</td>
			<td>Thomas scientific&#160;</td>
			<td>4663H25</td>
			<td style="width:444px;">alternatively, cut Corning Costar 3428 membranes off of transwell supports</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Instant glue</td>
			<td>purchase at hardware store</td>
			<td style="width:133px;">gel based preferrably</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 x 10 mm plates</td>
			<td>Falcon&#160;</td>
			<td style="width:133px;">351008</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7% agarose</td>
			<td>Sigma</td>
			<td>&#160;A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minutien pins</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:133px;">26002-20</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red free media (DMEM)</td>
			<td>Gibco&#160;</td>
			<td style="width:133px;">21063-029</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (100 mg/ml)</td>
			<td>AnaSed&#160;</td>
			<td style="width:133px;">139-236</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Matrigel</td>
			<td>BD</td>
			<td style="width:133px;">356231</td>
			<td style="width:444px;">basement membrane matrix, growth factor reduced, phenol red-free</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-4% agarose</td>
			<td>Sigma&#160;</td>
			<td>A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging of fixed intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vaseline</td>
			<td style="width:223px;">purchase at pharmacy</td>
			<td></td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lanolin</td>
			<td style="width:223px;">Sigma&#160;</td>
			<td>L7387</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:223px;">Surgipath</td>
			<td>39601006</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1 x PBS</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Focus clear and Mount Clear</td>
			<td>CelExplorer Labs Co.</td>
			<td>&#160;F101-KIT</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling clay</td>
			<td>purchase at art supply store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double stick tape</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton applicator swabs</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic molds, 10mm x 10mm x 5 mm)</td>
			<td>Tissue Tek&#160;</td>
			<td>4565</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">slides</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lab wipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td style="width:444px;">lint free delicate task wipe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Theiler staging chart&#160;</td>
			<td></td>
			<td></td>
			<td style="width:444px;"><a href="http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf">http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5X confocal microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMI 6000 stand&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aqueous mounting medium (Prolong Gold)</td>
			<td>Molecular Probes&#160;</td>
			<td>P36930</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Costar</td>
			<td>&#160;3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td style="width:444px;">used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Goat serum</td>
			<td></td>
			<td></td>
			<td style="width:444px;">used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tween20&#160;</td>
			<td>Sigma</td>
			<td>&#160;P9416</td>
			<td style="width:444px;"></td>
		</tr>
	</tbody>
</table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

在胎儿小肠大部分形态过程已经推断出从固定的组织薄片，提供了发展阶段变化的快照。由薄的连续切片的三维信息可以是具有挑战性的，因为完美重构连续切片和保持组织在连续切片的适当定向的难度来解释。由格罗斯等人最近的研究结果，2011年重点在了解肠1的开发绒毛形态三维信息的重要性。单独使用标记的肠细胞的三维重建表明大多数肠上皮细胞的接触既心尖和基底表面。此外，肌动蛋白细胞骨架在上皮的顶端表面的三维重建表明肠腔是连续的，并且次要流明是第一个工件ectioning。这两点，伴随着interkinetic核移植的肠上皮示范，明确了发展中肠上皮为假复层上皮，不分层，如以前认为1。观察到上皮三维的能力是开创性到证明了这一点，重新定义上皮形态在胎儿肠。用多光子成像技术和三维重建软件的演化，能够可视化完好，显影机构正在迅速提高。双光子激发可损害较小穿透更深的组织具有较高的分辨率。双光子成像和三维重建整个胎鼠肠中的沃顿等人 ，2012帮助定义绒毛生长2的格局。在这里，我们描述了整个器官培养系统，使离体的发展是文化系统的绒毛和扩展，使肠道是三维的开发过程中进行成像。

胎儿肠的整个外植体的培养可为位置的信号分子的坐标肠的发展，分配，和持续时间的分析，因为该系统能够与药理学试剂或重组蛋白质的信令的操作。 Transwell小培养系统（ 图1A中 ，从沃尔顿等再现。，2012）2提供了一种空气-液体界面，这使得它能够与毒品或蛋白质浸泡琼脂糖珠放置到组织中，从而建立本地信令资源（ 图1B ）。肠子从胚胎E10到E18....

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

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 ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

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

14.8K 次观看.  University of Michigan. 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. 

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

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

老鼠胎儿整肠文化系统

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