We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

注：动物实验均按照健康指南的国家研究院​​实验动物的护理和使用进行。委员会对日本东北大学医学院动物实验批准本文所述的实验步骤。 1，麻醉和开腹手术<ol style="margin-left: 40px;"><li>采血，用无特定病原体雄性SD大鼠。快速大鼠至少18小时，同时提供水。采集的血液用退休种鸡雄性大鼠在6-8个月的婴儿（550-650克）。 </li><li>流2.5-4.0％异氟醚，其是吸入麻醉剂，成箱连接到麻醉装置引入麻醉在2.0-3.5升/分10分钟。大鼠转成箱地介绍麻醉。 <ol><li>通过检查该响应的刺激与钳爪的损失确认麻醉。覆盖无线鼠的鼻子TH连接到麻醉设备，采血过程中深深地麻醉大鼠口罩。的浓度和流量维持在2.5-4.0％和2.0-3.5升/分钟，分别。 注意：氟烷不应被用于吸入麻醉，因为胚胎培养是从大鼠麻醉，该试剂显示出在胚胎发育的延迟相比，从胚胎大鼠用异氟烷麻醉22中得到的血清中培养收集的血清中。 </li></ol></li><li>为了避免毛皮的污染，消毒皮肤浇70％乙醇到麻醉的大鼠的腹部。拿起消毒皮肤和腹壁在用一对钳子的下部区域并同时切割这些层朝向胸部区域具有对大剪刀，以暴露内部器官。反映肠道腹腔有一对钳子的外面。 </li><li>切开皮肤和腹壁向后肢用一双大剪刀进一步暴露后腹部。反映了多余的脂肪在腹腔有一对钳子的外面。 </li><li>拿起内脏脂肪在使用两对钳子的中线，并仔细在一个平行的方向分割的脂肪，以暴露腹主动脉。重复此过程，以分裂内脏脂肪在纵向上可以轻松地找到腹主动脉分叉处。此外，主动脉是搏动，并且可以使用这种特性的相邻腔静脉进行区分。 </li><li>拿起腹主动脉周围脂肪和分叉小心地用两对钳，分离脂肪在腹主动脉。 </li></ol> 2，采血车<ol style="margin-left: 40px;"><li>小心地插入一21号针头连接到20毫升注射器立即颅内主动脉分叉处，以在向下的方向的斜面的尖端。握住注射器用一个手，拉注射器慢慢加收15毫升血液从雄性大鼠。 </li><li>从注射器取下针头，并倒入血成两个冰冷的10毫升无菌试管中（ 图1A）。保持在冰上的管中，直至离心延缓血液凝块的形成。离心管几个在同一时间减少离心时间。 </li><li>通过开胸手术和切割心脏或斩首安乐死的麻醉大鼠。 </li></ol> 3。大鼠血清的制备<ol style="margin-left: 40px;"><li>离心分离所采集的血液进行5分钟，在1200×g离心和室温（RT）到血液分离成上层和下层（ 图1C）。 </li><li>保持所述管，在4℃下进行1.5-2小时，以固定的纤维蛋白凝块。 </li><li>拿起纤维蛋白凝块中使用一对弯钳的上层，并且挤压的纤维蛋白凝块，分离血清。然后，按纤维蛋白凝块面临做的弯钳wnwards两个层（ 图1D）之间的界面附近。 </li><li>重复步骤3.3，以进一步分离纤维蛋白凝块。 </li><li>离心试管中5分钟，在1200×g离心和RT（ 图1E）。 </li><li>小心地在空气层流柜用无菌移液管将血清转移至15毫升无菌试管中。 </li><li>离心5分钟，收集的大鼠血清在1200×g离心及RT以除去任何剩余的红细胞。 </li><li>仔细普尔血清成新15毫升使用无菌移液管，以避免污染残余的红细胞在管（ 图1F）的底部灭菌试管中。 </li><li>孵育在水浴中的血清在56℃30分钟以灭活补体系统。 </li><li>冷却大鼠血清的试管至RT，在该空气层流柜中​​。该血清应当等分，以个别无菌试管10毫升存储该管在-20℃直到使用（ 图1H）。 </li></ol>

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No conflicts of interest are declared.

在WEC实验可重复性的结果是依赖于使用高品质IC大鼠血清和准确的胚胎解剖技术3。不要缩短采血前禁食期间，因为禁食是必要的血液中的葡萄糖浓度标准化。雌性大鼠不应该被用来进行血清的制备，因为激素水平在雌性动物按动情周期的变化，并且这种变化在雌二醇的激素不适用于胚胎培养物。此外，极其脂肪酸雄性大鼠不应该用于血清制备，以避免在血液中大量脂肪污染。 <p class="jo...

各种型号的动物被用于发育生物学在分子和细胞水平研究发育机制。例如，两栖类和鸟类物种已被广泛地用作传统的模型动物，都适合直接操纵的胚胎，因为这些胚胎发育母体之外。相对于这些动物，哺乳动物胚胎生长在母亲的子宫，并且在稍后阶段的增长是关键取决于子宫的功能。因此，它通常是难以直接操纵哺乳动物胚胎如那些来自小鼠，大鼠在早期阶段。在20世纪60年代，丹尼斯新成立的哺乳动物全胚胎培养（WEC）技术，使用WEC设备有连续供氧和热控制1。职业英语运动中，小鼠和大鼠胚胎能生长在体外 ，（ 即子宫外）。虽然WEC技术是通过添加各种CHEMIC常用于畸形人的化合物到培养基中，这种技术也被用在各种发育生物学研究，以了解在哺乳动物2-4独特发育机制。例如，WEC是通过使用荧光染料5，细胞移植6，并通过脂质转染7和电穿孔8-13基因导入结合其他技术，如细 ​​胞标记，在野生型和突变体胚胎。 最近， 在子宫内的操作已经被用于分析在啮齿动物胚胎的发育过程的后期和已结合电穿孔技术14-16。然而，这些技术不适合于由于难以在早期阶段实现精确局部注射的DNA溶液成胚胎的着床后和妊娠中期胚胎的操纵。虽然超声引导细胞移植和注射病毒载体的成早期胚胎（ 即，E8.5-E-9.5中的鼠标）， 在子宫内已有报道以前17,18，优秀的技能都需要有很高的成功率进行这些实验。因此，WEC与高可达体外具有优势对于小鼠和大鼠胚胎的操控。 从雄性大鼠制备立即离心（IC）的大鼠血清通常用于WEC介质。当胚培养在植入后阶段（ 即，早于E8.0在小鼠或E10.0大鼠），合成培养基和IC大鼠血清的混合物通常被用作用于WEC 19的介质。但是，要培养胚胎在妊娠中期（ 即 。，E8.0-12.5在小鼠胚胎或E10.0-E14.5大鼠胚胎），100％的血清应作为一个媒介，因为目前没有可用的替代介质允许胚胎在体外正常生长超过2天。 高品质的大鼠血清的制备是在WEC的实验实现重复性的关键一步。相比延迟离心，集成电路具有减少溶血时的血清被收集通过挤压纤维蛋白凝块，因为大部分的红血细胞已被分离的纤维蛋白凝块的好处。如溶血性大鼠血清不能支持大鼠和小鼠胚胎的正常生长，血清的使用即时离心制备优选使用延迟离心制备。我们的协议包含其他协议相比，两个额外的步骤18-20（ 即 ，，储存在冰上收集的血液前，先离心和保持所收集的血液样本，2小时，4℃第一次离心后）。前者步骤可延缓血液凝块的形成，而后者步骤促进纤维蛋白凝块，易于压缩的固化。因此，我们的协议可以由初学者。然而，重现采血及血清提取准确地单指协议的书籍是非常困难的19-21。在这个视频文章中，我们展示我们的标准协议，以获得最佳的IC大鼠血清，其中包括来自雄性大鼠和提取血清离心的腹主动脉采血的准备。

<table border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

图1示出的所描述的程序，用于血细胞和血清的分离代表性结果。我们一般会取得将15ml的血液从一个退休的雄性大鼠（ 图1A）。通过离心，收集的血液可以被分离成含有血清和血纤蛋白凝块，这是用箭头表示，并将含有血细胞（ 图1C）的下层的上层。重要的是，溶血的血样不能被分离成血清和血液的层（ 图1B）。离开该管在4℃下2小时后，血纤维...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.7K 次观看.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.