这项工作是由医学研究委员会的核心资金支持。我们感谢克雷格·尼科尔编制技术图纸。我们感谢马修·皮尔逊和保罗·佩里为他们的影像支持。

由英国内政部（项目许可证编号PPL三千七百八十五分之六十〇和四千四百二十四分之六十〇）按照许可制度的指引下进行的所有动物的工作。 1。准备<ol><li>通过结合荧光报道鼠标线适当Cre重组酶表达小鼠线标记在显影皮肤的黑素细胞。 酪氨酸:: CREA，酪氨酸:: CREB 8和WNT1 ::酶Cre 14已被成功地用作Cre的表达株系和R26YFPR 9当记者一行。 </li><li>准备在层流罩的培养基;补充的Dulbecco改良的Eagle培养基（DMEM）与（1％体积/体积青霉素/链霉素），10％体积/体积的胎牛血清和0.584克/ L的L-谷氨酰胺。 </li><li>实时成像设备在图1中概述了通过擦拭下用70％乙醇消毒成像室基部。由OV浸泡消毒刀片，影像剪辑，和O型圈ernight在70％乙醇中。从6孔板中使用的无菌盖的所述腔室的盖子。 </li><li>成像腔室的底部表面由通过一个O形环（ 图1）保持在适当位置一个lummox膜。先从35毫米透气性lummox菜。将培养皿在成像腔和切出的气体渗透膜用单刃剃刀片。将O型圈在膜将其固定到成像室。 </li><li>成像剪辑（共6个）夹紧胚胎皮肤到位（ 图1），他们必须充满1％琼脂糖后再使用。通过微波，冷却至60°C。准备琼脂糖用无菌DH 2 O的2％溶液混合1点01分，用10ml培养基（见步骤2）预热至60℃。站在6影像片段在无菌6孔板。滴落的1％琼脂糖溶液倒入用细pastette每个片段的中心，并允许冷却。加入1毫升冷的无菌PBS至各孔，以防止一从晒出garose。摄像剪辑可以存储几个小时的PBS中。 </li><li>开展胚胎皮肤的细腻分离和处理的解剖组织使用的头发粘法“作为柏木等 3所描述而不是使用筷子竹烤肉串可以使用。做一个小切口在使用单刃刀片的扦子的平端插入一个柔软的牙刷的刷毛。修剪所述扦子，以便它的长度为15cm左右。 </li><li>使用共聚焦显微镜采用了全环境 ​​室或舞台顶室提供5％CO 2的空气中。 Prewarm显微镜载物台至37℃至少1小时前成像。这有助于防止因膨胀阶段元器件的样本漂移，因为它们升温。 </li><li>使用显微镜的控制软件准备了多套位置，这样一方面可以快速，轻松地集中在每一种文化的中间设立一个experimen万吨。 </li></ol> 2。程序<ol><li>交配的小鼠和指定的第一天交配后的一天E0.5。在E14.5天，通过颈脱位法宰杀阴和通过一个小的中线切口，拉开朝向头部和尾部的皮肤，然后切割底层腹膜和敲打回到身体的两侧暴露的体腔。解剖子宫放入冷水无菌PBS用钳子夹住坚决和使用手术剪沿系膜及剪断。保留子宫置于冰上，直到需要。有关详细说明见Shea 等。（2007）15。 </li><li>通过首先切割子宫壁与外科剪刀的长度，以释放蜕膜然后解剖从蜕膜的胚胎通过它们拉开有两对细镊子解剖从子宫胚胎。屏幕为荧光报道表达式（如果需要）。 </li><li>前宰杀解剖胚胎斩首。替补equently拆下尾，并用单刃剃刀片后肢然后前肢。如果需要保留的组织进行基因分型。 </li><li>使在ventrum靠近中线的rostro脚方向的切口。用卷发棒在步骤1.6中所述，小心地挑逗远离底层的结缔组织的同时转动胚胎皮肤。不要完全去除皮肤，而是让它沿着从第一切口腹缘最远连接。 </li><li>重要的是不要让皮肤变得搞砸了，因为它是那么很难安装是非常重要的。为了防止这种情况，用卷发棒（皮肤的一面朝上），干燥的培养皿的表面变平的皮肤，然后切离胚胎躯干使用单刃刀片。真皮侧可以从表皮侧通过仔细观察的解剖组织和胚胎之间的交界处被识别的皮肤被移除之前。的解剖组织的面积为大约0.5mm 2的解剖胚胎E14.5时。 </li><li>放置成像夹在皮肤上的顶部，以使琼脂糖接触到组织的真皮侧。静置60秒，然后小心地挑逗组织了夹子的两侧边缘。颠倒的剪辑，并使用发粘变平的皮肤和除去任何气泡，小心触摸到组织的表面（在将要成像的面积）尽可能少。 </li><li>用丝线缝合线用两个简单的拇指结对成像夹两侧的皮肤绑成像片段，让他们拧紧和拉扯皮肤进沟接近剪辑的开头。颠倒的片段，推成像插入并装在基座内。用约5ml培养基的填充插入件（参见步骤1.2）。 图1概括成像室的组装。 </li><li>重复协议的其余5个样品。 </li></ol> 3，活成像<ol><li>成像腔室具有相同的尺寸为标准6孔板中。使用适当的板插入到安装在室内的共聚焦显微镜的阶段。 </li><li>一种自动化的阶段是必需的，以图像中的平行的培养物。定义了六个位置到图像中的共聚焦软件。确切的设置将取决于所使用的显微镜​​的能力。通常是一个小的z栈（3-5 z轴位置以考虑任何阶段漂移）用于在每一个6位的和的z栈被捕获每2分钟为18-24小时。用于活成像应用中，通常有利的是使用一种比旋光最佳针孔设定较宽，从而降低激光器功率和所需的z部分的数目。 </li></ol>

<ol>
	<li>Thomas, A. J., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+a+melanocyte:+the+specification+of+melanoblasts+from+the+neural+crest.">The making of a melanocyte: the specification of melanoblasts from the neural crest.</a> Pigment Cell &amp; Melanoma Research. 21 (6), 598-610 (2008).</li><li>Mayer, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migratory+pathway+of+neural+crest+cells+into+the+skin+of+mouse+embryos.">The migratory pathway of neural crest cells into the skin of mouse embryos.</a> Developmental Biology. 34 (1), 39-46 (1973).</li><li>Kashiwagi, M., Huh, N., Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture+of+Developing+Mouse+Skin+and+Its+Application+for+Molecular+Mechanistic+Studies+of+Morphogenesis.">Organ Culture of Developing Mouse Skin and Its Application for Molecular Mechanistic Studies of Morphogenesis.</a> Epidermal Cells: Methods and Protocols. 289, 39-45 (2005).</li><li>Kashiwagi, M., Kuroki, T., Huh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+hair+follicle+formation+by+epidermal+growth+factor+in+an+organ+culture+of+developing+mouse+skin.">Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.</a> Developmental Biology. 189 (1), 22-32 (1997).</li><li>Van Der Wel, L. I., Wei, L., Prens, E. P., Laman, J. D., Companjen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+ex+vivo+skin+organ+culture+system+for+functional+studies.">A modified ex vivo skin organ culture system for functional studies.</a> Archives of Dermatological Research. 293 (4), 184-190 (2001).</li><li>Mort, R. L., Hay, L., Jackson, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+live+imaging+of+melanoblast+migration+in+embryonic+mouse+skin.">Ex vivo live imaging of melanoblast migration in embryonic mouse skin.</a> Pigment Cell &amp; Melanoma Research. 23 (2), 299-301 (2010).</li><li>Jordan, S. A. A., Jackson, I. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGF+(KIT+ligand)+is+a+chemokinetic+factor+for+melanoblast+migration+into+hair+follicles.">MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles.</a> Developmental Biology. 225 (2), 424-436 (2000).</li><li>Delmas, V., Martinozzi, S., Bourgeois, Y., Holzenberger, M., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-mediated+recombination+in+the+skin+melanocyte+lineage.">Cre-mediated recombination in the skin melanocyte lineage.</a> Genesis. 36 (2), 73-80 (2003).</li><li>Srinivas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1 (1), (2001).</li><li>Li, 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=Rac1+Drives+Melanoblast+Organization+during+Mouse+Development+by+Orchestrating+Pseudopod.">Rac1 Drives Melanoblast Organization during Mouse Development by Orchestrating Pseudopod.</a> Driven Motility and Cell-Cycle Progression. Developmental Cell. 21 (4), 722-734 (2011).</li><li>Li, 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=Activated+Mutant+NRas(Q61K)+Drives+Aberrant+Melanocyte+Signaling,+Survival,+and+Invasiveness+via+a+Rac1-Dependent+Mechanism.">Activated Mutant NRas(Q61K) Drives Aberrant Melanocyte Signaling, Survival, and Invasiveness via a Rac1-Dependent Mechanism.</a> The Journal of Investigative Dermatology. , 1-12 (2012).</li><li>Schachtner, H., 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=Tissue+inducible+Lifeact+expression+allows+visualization+of+actin+dynamics+in+vivo+and+ex+vivo.">Tissue inducible Lifeact expression allows visualization of actin dynamics in vivo and ex vivo.</a> European Journal of Cell Biology. 91 (11-12), 923-929 (2012).</li><li>Ma, Y., 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=Fascin+1+is+transiently+expressed+in+mouse+melanoblasts+during+development+and+promotes+migration+and+proliferation.">Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation.</a> Development. 140 (10), 2203-2211 (2013).</li><li>Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., 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=Modification+of+gene+activity+in+mouse+embryos+in+utero+by+a+tamoxifen-inducible+form+of+Cre+recombinase.">Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.</a> Current Biology. 8 (24), 1323-1326 (1998).</li><li>Shea, K., Geijsen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+6.5+dpc+mouse+embryos.">Dissection of 6.5 dpc mouse embryos.</a> Journal of Visualized Experiments. (2), (2007).</li></ol>

作者什么都没有透露。

我们描述一个方法来培养胚胎的皮肤是特殊性服从活细胞成像的共聚焦倒置显微镜。该方法包括近期的改进，允许6文化进行成像的并行和消除了对基质胶与原方法的6核孔膜的依赖。从类似的技术的关键技术的区别在于使用具有透气性的lummox膜的建立空气液体界面并且还充当盖玻片。我们已经成功地保持了连续的共焦成像的培养长达48小时。高分辨率和出色的信号发送到共焦图象的信噪比?...

传统胚胎皮肤已经培养了来自小鼠胚胎解剖和安装在聚碳酸酯核孔膜。然后将膜漂浮在培养基中，从而保持一个气-液界面在发展中国家组织3,4的表面上。这一技术已被用于固定组织，并评估用β-半乳糖苷酶作为标记7黑素细胞分布测定黑素细胞的行为。我们已经开发出一种方法，可以让在体外培养皮肤的6个现场成像的荧光标记的黑素细胞。在这里，我们描述的方法在细节上剥离，设置，住聚焦成像，其中包括最近的一些改进。 黑素细胞是黑素细胞的胚胎前体，产生色素在头发和皮肤细胞。黑素细胞起源在邻近神经管的神经嵴在胚胎周围每天9（E9）在发育中的小鼠émbryo。随后，他们沿着外胚层和开发体节之间的背侧途径迁移。在E12.5他们从真皮层移动到它们增殖并继续他们的迁移表皮。初级毛囊格局开始形成本地化是这些毛囊在表皮在E14.5和E15.5黑素细胞。对于黑素细胞/黑素细胞发展的综述参见托马斯＆埃里克森（2008年）1。以标签的黑素细胞的人口，我们结合酪氨酸::动物CREB表达Cre重组酶的小鼠酪氨酸酶启动子8与R26YFPR动物表达黄色荧光蛋白（YFP）有条件地从ROSA26轨迹9驱动。 我们描述的方法来使用倒置共聚焦显微镜培养胚胎皮肤和采集图像。它适于形式在莫特等人描述的原始方法（2010）6。本方法使成像在6孔格式和消除了对核孔膜和基质胶上的依赖，以支持培养。代替用1％琼脂糖的一个小块，以稳定胚胎皮肤。去除基质胶上的依赖是很重要的特别是当胚胎皮肤的可溶性生长因子的响应是研究的重点。我们原来的方法已经被用来获得新的见解黑素细胞发展10-13和我们预期，我们在这里描述的改进将使其更加强大的实验技术特别是在多个并行的文化是一个要求。

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM high glucose</td>
			<td style="width:132px;">Biochrom AG</td>
			<td style="width:119px;">F0475</td>
			<td style="width:167px;">without phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">P3032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">S9137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:132px;">Hyclone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Live imaging chamber</td>
			<td style="width:132px;">Custom made</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lummox dishes</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-well plate</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">657-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single edged razor blade</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">1244-3170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:132px;">Biogene</td>
			<td style="width:119px;">300-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine pastette</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kebab skewers</td>
			<td style="width:132px;">Waitrose</td>
			<td style="width:119px;"></td>
			<td>Bamboo BBQ skewers 30cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toothbrush</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Suture thread</td>
			<td style="width:132px;">Look</td>
			<td style="width:119px;">SP115</td>
			<td>Black silk suture thread</td>
		</tr>
	</tbody>
</table>

ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration

黑素细胞是黑素细胞衍生的前体神经嵴;细胞负责产生色素在皮肤和头发。黑素细胞经过胚胎，他们随后殖民开发毛囊1,2的表皮迁移。神经嵴细胞的迁移是广泛的研究在体外 ，但体内的方法仍然没有得到很好的发展，特别是在哺乳动物系统。一种替代方法是使用体外器官型培养3-6。小鼠胚胎皮肤培养需要一个气-液界面（ALI）的跨组织3,6的表面的维护。高分辨率实时成像的小鼠胚胎的皮肤已经受到阻碍，缺乏一个很好的方法，它不仅保持了这个ALI的也可以让文化被倒置，因此，短工作距离物镜，最共聚焦显微镜兼容的。本文介绍了最新改进的甲基OD使用的气体透过膜，以克服这些问题，并允许胚胎皮肤中体外培养6的高分辨率共焦成像。通过使用黑素细胞特异性表达Cre重组酶表达小鼠线结合R26YFPR记者一行，我们能够这些皮肤文化中，以荧光标记的黑素细胞的人口。该技术允许实时成像黑素细胞，并观察他们的行为，并与他们发展组织的相互作用。代表性的成果包括以展示能力，生活图像6文化并行。

图2显示了从一个时间间隔实验使用来自酪氨酸胚胎皮肤代表性结果:: CREB x R26YFPR胚胎在E14.5。 6胚胎皮肤培养物的共聚焦显微镜成像，每2分钟18小时。免费软件图像分析软件包的ImageJ用于分析在6电影的YFP标记的黑素细胞的行为。在黑素细胞是使用wrMTrck插件由帕尼SøndergaardPedersen的开发（自动跟踪<a href="http://www.phage.dk/plugins/wrmtrck.html">http://www.phage.dk/plugins/wrmtrck.html</a> ）?...

Watch this Scientific Journal Video about Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration at JoVE.com

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

我们描述了小鼠胚胎皮肤的解剖和体外培养。培养系统保持一个气 - 液界面在整个组织表面，并允许成像于倒置显微镜。黑素细胞中，显影皮肤的成分，被荧光标记的允许使用共聚焦显微镜观察其行为。

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 ...

ex-vivo-culture-mouse-embryonic-skin-live-imaging-melanoblast

Research

JoVE Journal

Biology

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

12.5K 次观看.  University of Edinburgh. 我们描述了小鼠胚胎皮肤的解剖和体外培养。培养系统保持一个气 - 液界面在整个组织表面，并允许成像于倒置显微镜。黑素细胞中，显影皮肤的成分，被荧光标记的允许使用共聚焦显微镜观察其行为。 

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

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

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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 ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...