This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

所有的动物实验和程序由美国杜兰大学的机构动物护理和使用委员会（IACUC）的批准。 1.手术过程设置<ol><li>高压灭菌仪器，手术用品，并在手术前文化用品。每只大鼠手术耗材包括:1悬垂性好，悬垂1与预切孔（0.5英寸×1.5英寸）的中心，纱布垫，1个吸水焊。手术器械包括:1手术刀用10号刀片，2双镊子和一对细剪刀。培养用品包括:1的悬垂性，1镊子，并准备用的聚碳酸酯过滤器6孔板插入。 </li><li>消毒一个有机玻璃平台，手术阶段和用70％乙醇的外科工作台空间。保持手术分期在无菌碗中待用。 <ol><li>在100毫米培养皿的中心钻孔在大约2通过1创建一个手术阶段。接下来，用砂纸顺利任何尖锐的边缘，并添加硅胶胶水层到孔的边缘，以创建用于组织中升高的表面。 </li><li>另外，采用CAD软件设计手术阶段和3-D打印（ 图1）做出。 </li></ol></li><li>将无菌吸水焊下来，躺在它上面一个有机玻璃平台。放置悬垂未经预切孔，在旁边的吸湿焊加热垫。 </li><li>预暖的无菌磷酸盐缓冲盐水（PBS），介质和盐水至37℃。地方媒体和PBS单独培养皿在50mL锥形旁边的外科设置管加热垫和地方盐水顶上。 </li><li>确保所有软件包之前，手术开始打开，确保所有材料的消毒处理。在此过程中使用的常用工具的完整列表中列出 具体的手术材料和工具表 。 </li></ol> 2. Mesenterÿ组织收获<ol><li>使用成年雄性Wistar大鼠（350±25克; 6 - 8周龄）。其它菌株和大鼠的年龄可以被取代。 </li><li> 通过氯胺酮（80毫克/公斤体重）和赛拉嗪（8毫克/公斤体重）的肌内注射麻醉大鼠。确认大鼠处于麻醉状态的脚趾之间掐来检查反射反应;应该没有。此终端过程先发制人镇痛是没有必要的。 </li><li>剃须腹部和使用脱毛膏去除剩余的头发。用70％的异丙醇接着碘伏擦拭腹部皮肤的两倍。用于擦拭物的外科医生应该开始在手术部位的中心，并移动到圆形方式制备的区域的外侧，以不重叠的那些具有相同的一块无菌纱布或无菌棉签预先洗涤的区域。然后动物转移到无菌手术的设置，然后将有机玻璃高原之上形成。 </li><li>使用手术刀刀片，使在一个0.75 - 1.25在切口在胸骨下方开始1肠道。小心不要刺破肠或肠系膜（1层皮肤，1层结缔组织，1层肌肉）。 </li><li>放置一个悬垂超过切口一个预切孔和放置无菌手术阶段悬垂的顶上。确保与切口对齐开幕。用消毒棉签涂抹定位，并通过手术分期开盘拉出回肠。 </li><li>用棉签涂抹通过阶段8肠系膜窗口，并注意不要触碰窗口（ 图1） -拉6。组织通常由小肠盲肠附近开始的回肠区域采收。保持暴露组织湿润使用无菌注射器滴下的溶液根据需要加热的无菌盐水。 </li><li>安乐死通过戊巴比妥钠的心内注射鼠（每只鼠0.2mL）中。在卸下我senteric窗口，确保老鼠被触诊心脏安乐死;应该没有脉冲。 </li><li>通过使用镊子夹住脂肪垫和精细剪刀剪开窗口中删除所需的肠系膜组织。离开的脂肪（2mm）的窗口周围的边框。媒体在温暖无菌PBS洗一次组织一次。 </li><li>返回形象化回肠到腹腔，并根据机构指南动物的处理。 </li></ol> 3.肠系膜组织培养时间推移研究<ol><li>传输蒸压文化用品（参见1.1节）和组织无菌层流罩。 </li><li>用镊子每个组织转移聚碳酸酯过滤膜上面。由脂肪垫抓组织，以避免损坏血管。 </li><li>使用快速脂肪垫，小心不要碰到窗口传播组织。倒置与组织插入到6孔板的底部，并用3毫升培养基（ 图1覆盖</st荣&gt;）。用于这一过程的典型介质包括极限必需培养基（MEM）中，用1％青霉素链霉素（PenStrep）和10％胎牛血清（FBS）。媒体可辅以其它血清和/或生长因子刺激血管生成。 </li><li>重复步骤3.2 - 3.3长达5天的标准培养箱条件的每个组织和培养（5％CO 2，37℃）。 </li></ol> 4.肠系膜组织的时间推移成像<ol><li>上成像的当天，用共轭BSI-凝集素补充媒体在每个孔和标准培养条件下温育30分钟。提供免费凝集素媒体组织洗净两次。 BSI-凝集素染色将肠系膜组织保持可见在培养3天。 </li><li>板块转移到显微镜阶段。标识基于其形态和网络结构的血液和淋巴管。 </li><li>找到每个组织所需的网络区域，并拍摄图像。记下成像位置，以确保在同一区域将被捕获为后续图像。如果使用机动阶段，记录的坐标。 </li><li>返回组织的孵化器，并继续培养至所需的终点。根据所需实验时间点需要4.3 - 重复步骤4.1。 </li></ol> 5.组织免疫标记<ol><li> BSI-凝集素标签 <ol><li>在37°C孵育组织30分钟，在媒体后跟两个漂洗与媒体1:40 FITC-缀合凝集素（在6孔板每孔2.5毫升抗体溶液）。对于漂洗，添加媒体，然后立即更换。 </li></ol></li><li> 活/死贴标 <ol><li>在37℃下用1孵育组织10分钟:500 2毫乙锭同型二聚体-1和1:500 1毫钙黄绿素中媒体后跟两个漂洗用媒体的AM（在6孔板每孔2.5毫升抗体溶液）。 </li></ol></li><li> BSI-凝集素/ NG2标签 <ol><li>在显微镜传播组织下滑E（1 - 2组织/幻灯片），并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。用PBS（3×10分钟）冲洗组织。 </li><li>初级抗体标记孵育1在室温下1小时的组织:100兔多克隆NG2抗体和5％正常山羊血清（NGS）。用PBS（3×10分钟）冲洗组织。 </li><li>对于二级抗体标记孵育在室温下1小时的组织以1:100的山羊抗 - 兔CY2共轭抗体（GAR-CY 2）和5％NGS。用PBS（3×10分钟）冲洗组织。 </li><li>孵育在室温下30分钟，组织用PBS中1:40 FITC-缀合凝集素后跟两个漂洗用PBS。对于漂洗，加入PBS，然后立即更换。 </li><li>要安装的幻灯片，覆盖在上面以50:50 PBS组织和甘油溶液和地点盖玻片。使用密封指甲油幻灯片边缘。 </li></ol></li><li> LYVE-1 / PECAM贴标<ol><li>铺在显微镜载玻片组织（1 - 2组织/滑动）并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li> 200小鼠单克隆生物素化的CD31抗体和1:初级抗体标记在室温下用1孵育1小时组织100兔多克隆LYVE-1的PBS + 0.1％皂角苷+ 2％牛血清白蛋白抗体（BSA）+ 5％NGS 。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>对于二级抗体标记，孵育组织为在室温下用1 1小时:500 CY3共轭链霉抗抗体和1:在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS 100 GAR-CY2。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>要挂载幻灯片，封面组织以50:50 PBS和甘油溶液并放置一个盖玻片放在上面。使用密封指甲油幻灯片边缘。 </li></ol></li><li> 的BrdU / BSI-凝集素标签 <ol><li> 1毫克/毫升的BrdU添加到媒体并用BrdU溶液替换组织媒体。在37℃下孵育2小时。 </li><li>铺在显微镜载玻片组织（1 - 2组织/滑动）并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。用PBS（3×10分钟）冲洗组织。 </li><li>变性组织的DNA中的2M HCl中在37℃下1小时。在PBS + 0.1％皂角苷（3×10分钟）冲洗组织。 </li><li>初级抗体标记，孵育组织为在室温下用1 1小时:100单克隆小鼠抗BrdU在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>对于二级抗体标记，孵育在室温下1小时的组织以1:100的山羊抗小鼠的Cy3缀合的抗体（GAM-Cy3标记）在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><lI&gt;孵育在室温下30分钟，组织用PBS中1:40 FITC-缀合凝集素后跟两个漂洗用PBS。 </li><li>要安装在上面滑动，封面组织以50:50 PBS和甘油溶液和地点盖玻片。使用密封指甲油幻灯片边缘。 </li></ol></li><li> BSI-凝集素/ CD11b的标签 <ol><li>铺在显微镜载玻片组织（1 - 2组织/滑动）并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>初级抗体标记孵育在室温下1小时的组织以1:100的小鼠抗大鼠的CD11b在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>对于二级抗体标记孵育在室温下用1 1小时组织:100 GAM-Cy3的在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。用PBS洗涤组织+ 0.1％皂角苷（3×10分钟）。 </li><li>孵育在室温下30分钟，组织用PBS中1:40 FITC-缀合凝集素后跟两个漂洗用PBS。 </li><li>要安装在上面滑动，封面组织以50:50 PBS和甘油溶液和地点盖玻片。使用密封指甲油幻灯片边缘。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

该协议记录了使用大鼠肠系膜文化模型作为微血管网络增长的时间推移成像的体外工具的方法。在我们的实验室以前的工作已经建立了使用我们的1模型）血管生成8,2）淋巴管8，3）周细胞-内皮细胞相互作用8，和4）抗血管生成药物测试的9。对于在多个时间点培养的大鼠肠系膜组织成像的能力提供了用于评价组织特异性...

微血管网络的增长和重塑是用于组织功能共同点，伤口愈合，和多个病症和一个关键过程是血管生成，定义为新血管从已有的1，2的生长。组织工程新船或设计基于血管生成疗法，了解参与血管生成的细胞动力学的重要性是至关重要的。然而，这个过程是复杂的。它可以在一个微血管网络内的特定位置而变化，并且涉及多种细胞类型（ 即内皮细胞，平滑肌细胞，周细胞，巨噬细胞，干细胞）和多个系统（淋巴网络和神经网络）。虽然在体外模型已作出了巨大贡献，以检查参与血管生成3种不同的细胞之间的关系，它们的生理相关性可由于兵卫被削弱<html> - [R复杂性有限，事实上，他们没有真实地反映体内的情况。为了克服这些限制，三维培养系统3， 离体组织模型4中 ，微流体系统5，6，和计算模型7已经被开发并引入在最近几年。然而，仍然需要具有时间推移能力的模型来研究在完整微血管网络体外血管生成。血管生成研究建立新的延时模式与复杂性的水平将提供一个宝贵的工具来了解调节血管生成的基本机制，以提高治疗。 使血管跨越一个完整的微血管网络体外调查一个潜在的模型大鼠肠系膜养殖模式&gt; 8。在最近的工作中，我们已经表明，血液和淋巴微血管网络保持培养后存活。更重要的是，大鼠肠系膜培养模型可用于研究官能周细胞 - 内皮细胞相互作用，血液和淋巴管内皮细胞的连接，和延时成像。本文的目的是为我们的时间推移成像方法的协议。我们的代表性的结果文件证明血管生成的刺激与血清后保持存活，并提供使用用于量化组织特定的血管生成反应以及内皮细胞追踪研究该方法的实施例的多种细胞类型。

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			<td height="34" style="height:34px;width:165px;">Drape</td>
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			<td style="width:132px;">4012</td>
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			<td style="width:183px;">Roboz Surgical Instrument</td>
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			Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2&#34; Overall Length</td>
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			<td>FisherBrand</td>
			<td>13-761-52</td>
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			<td>Medical Action Industries Inc.</td>
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			<td style="width:183px;">LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td>
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			<td>Invitrogen</td>
			<td>14040-133</td>
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			<td>11095080</td>
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			<td>Invitrogen</td>
			<td>15140-122</td>
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			<td>Invitrogen</td>
			<td>16000-044</td>
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			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
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			<td height="21" style="height:21px;width:165px;">Povidone-Iodine</td>
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			<td>SIGMA</td>
			<td>320331</td>
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			<td>Fisher Scientific</td>
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			<td>AB5320</td>
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			<td height="21" style="height:21px;">PECAM (CD31) Antibody</td>
			<td>BD Biosciences</td>
			<td>555026</td>
			<td></td>
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			<td height="21" style="height:21px;">LYVE-1 Antibody</td>
			<td>AngioBio Co.</td>
			<td>11-034</td>
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			<td height="21" style="height:21px;">Goat Anti-Rabbit Cy2-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-585-144</td>
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			<td height="21" style="height:21px;">Goat Anti-Mouse Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-227-003</td>
			<td></td>
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			<td height="21" style="height:21px;">Streptavidin Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
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			<td>Invitrogen</td>
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			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
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			<td height="21" style="height:21px;">5-Bromo-2&#39;-Deoxyuridine</td>
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			<td>B5002</td>
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			<td height="52" style="height:52px;width:165px;">Monoclonal Mouse Anti-Bromodeoxyuridine&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Clone Bu20a</td>
			<td>Dako</td>
			<td style="width:132px;">M074401-8</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Rat CD11b&#160;</td>
			<td>AbD Serotec</td>
			<td>MCA275R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

血管生成，定义为新的血管从预先存在的血管的生长，包括内皮细胞，周细胞，平滑肌细胞，免疫细胞，并与淋巴管和神经的协调。多小区，多系统的相互作用必要的血管生成在生理相关环境的调查。因此，虽然使用体外细胞培养模型提供了机械的见解，一个共同的批评是它们不概括与微血管网络相关联的复杂性。该协议的目的是证明之前和在培养的大鼠肠系膜组织血管生成的刺激后，使完整的微血管网络的时移进行比较的能力。培养组织含有维持其层次微血管网络。免疫标记证实了内皮细胞，平滑肌细胞，周细胞，血管和淋巴管的存在。在一个ddition，与BSI-凝集素标记的组织使前后血清或生长因子刺激由毛细血管发芽和血管密度后，本地网络区域的延时比较。相比于普通细胞培养模型，这种方法提供了在生理学相关微血管网络内皮细胞谱系研究和组织特异性血管生成药物评价的工具。

后在培养3天后，组织被标记有活/死活力/细胞毒性试剂盒以证明在大鼠肠系膜培养模型（ 图2A）微血管的生存能力。大部分存在于肠系膜细胞保持在血管内皮细胞，根据他们在微血管段位置确定的文化活力。内皮细胞的增殖也被外源凝集素/ BrdU标记（ 图2D）确认。平滑肌细胞和周细胞的存在沿血管被证实与NG2标记（ 图2B）。标记为LYVE...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

an-ex-vivo-method-for-time-lapse-imaging-cultured-rat-mesenteric

Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K 次观看.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

视频: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

啮齿类动物胎盘的体外灌注

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

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