The University Paris 13 supported this work. ASPT was supported by a University Paris 13 fellowship and the "Association Fran&#231;aise pour le Syndrome d'Ondine". FJ was supported by a Laboratory of Excellence GR-Ex fellowship. The GR-Ex (ref ANR-11-LABX-0051) is funded by the program "Investissement d'avenir" of the French National Research agency (ref ANR-11-IDEX-0005-02).

注:C-FOS检测是涉及几个步骤（ 图1）的标准化过程。所有实验在大鼠或小鼠中进行。实验方案由伦理委员会在动物实验查尔斯·达尔文（CE5 / 2011/05）批准，按照2010年9月22日（2010/63 / EU）动物保健，欧洲共同体理事会指令完成，按照实施与法国的法律对动物的照顾。 1.溶液的制备<ol><li>制备磷酸盐的0.2M磷酸钠缓冲液:补充6.24克的NaH 2 PO 4 * 2H 2 O和22.56克Na 2 HPO 4 * H 2 O至1升蒸馏水，同时搅拌。 </li><li>准备4％多聚甲醛（PFA）:添加20克PFA的蒸馏水150毫升加热该溶液至80℃，同时搅拌。要清除溶液，减少热量并添加1N的NaOH的1或2滴，以帮助多聚甲醛溶解。完成至250毫升用蒸馏水和添加250毫升0.2M磷酸缓冲液（pH 7.4）。保存在4℃。使用这些试剂时，采取适当的照顾。带着手套，实验室外套，并且在化学罩护目镜处理。 注:在灌注前一天，准备4％PFA。确定4％PFA的体积的年龄，数目和动物物种的功能。 </li><li>制备冷冻保护溶液:在250ml的0.2M磷酸盐缓冲盐水中，混合5克聚乙烯吡咯烷酮，150克蔗糖和2.5克氯化钠。完全溶解后，增加150个毫升乙二醇的并调整到500毫升蒸馏水。储存在4℃的冷冻保护溶液。 </li><li>制备0.1M磷酸盐缓冲盐水（PBS）:加入18克NaCl的1升蒸馏水，同时搅拌。完全溶解后，加入磷酸钠缓冲液1升的0.2M。 </li><li>准备磷酸盐缓冲液添加0.3％的Triton X100（PBST）:加入3毫升三硝基甲苯X10的0 997毫升的PBS。 </li><li>制备的0.05M Tris-缓冲液（pH值为7.6）:添加3.03克的Tris-盐酸和0.70克亚磷酸三碱基至500ml，同时搅拌蒸馏水。 </li><li>准备2％山羊血清在PBST:一盘，准备加入30ml的解决方案。加入600微升山羊血清的至30ml PBST拌匀。加入每孔2.5毫升。 注意:使用血清必须来自用于二级抗体的产生种类</li><li>制备的兔多克隆抗体对c-fos蛋白（1:4000，SC-52）在PBST用牛血清白蛋白（0.25％）。对于一个盘子，准备加入30ml的解决方案。 75毫克牛血清白蛋白添加至30ml PBST拌匀。然后，添加7.5微升兔多克隆抗体对c-fos蛋白。 </li><li>制备生物素化的山羊抗兔抗体（1:500）在PBST与血清白蛋白的牛（0.25％）。对于一个盘子，准备加入30ml的解决方案。 75毫克牛血清白蛋白添加至30ml PBST拌匀。然后，添加60微升生物素化的山羊抗兔抗体。 </li><li>制备抗生物素蛋白 - 生物素 - 过氧化物酶复合物（1:250）在PBST（ABC溶液）。对于一个盘子，准备加入30ml的解决方案。加入试剂A的120微升和120微升试剂B〜30毫升PBST拌匀。 注:ABC的解决方案必须准备使用前至少30分钟。 </li><li>准备底物溶液。在即将使用之前，如下（为一个板）制备的溶液:添加20毫克硫酸镍铵和10mg 3.3二氨基四盐酸的至50ml Tris-缓冲液。过滤和避光。加入每孔2.0毫​​升。在剩余溶液（25毫升）加H 17微升2 O 2（H 2 O 2溶液H中2 O 30％）。加入每孔2.0毫​​升。 注意:适当的照顾应该用这些试剂时服用。手套和白大褂处理。 </li></ol> 2. c-fos INDUction和组织处理体内 注意:实验在大鼠（只SD）或小鼠（C57BL / 6）进行的。 <ol><li>放置的动物中有缺氧（O 2 8％的平衡N 2）或高二氧化碳（CO 2 4％，O 2 21％的平衡N 2）的气体混合物2小时（ 图2A）通风一个气密箱。 </li><li>麻醉用戊巴比妥腹膜内注射（100毫克/千克）动物。灌注动物transcardially用4％PFA 20。下一个化学罩护理和使用手套处理。 </li><li>灌注后，解剖脑20和浸入它在7ml 4％PFA中15毫升管在4℃下48小时。然后，从PFA取出大脑，并在冷冻保护溶液完全浸没了。商店在-18℃（ 图3）。由相应的废物处理处置PA动物的身体的其余部分的thway。 注意:冷冻保护是PFA固定和冷冻之间的工序。它减少了与其冷冻将是无定形形式的溶液冰晶的细胞的形成。在固定的样品必须在冷冻保护溶液（在步骤1.3中制备）为至少24小时，在4℃下进行浸渍。下化学罩护理和手套处理。 </li></ol> 3，c-fos诱导和组织处理离体 注意:实验在只新生大鼠或小鼠中进行。 <ol><li>如Suzue 19描述隔离中枢神经系统。放置在一个录音室，不断地与人工脑脊髓液在7.5毫升/分钟的速度在26°C 21（学联superfuse它:在MM:130.0氯化钠，5.4氯化钾，0.8 氯化钙 ，1.0 氯化镁 ，26.0的NaHCO 3，30.0 D-葡萄糖;用95％ 的 O 2和5放气饱和的 O 2，并调节至pH 7.4的％的CO 2）（ 图2B）。 </li><li>与低氧或通过酸化脑脊液（标准脑脊液用NaHCO 3降低到10.0毫脱氧脑脊液（用95％的N 2和5％的CO 2，pH 7.4的鼓泡相同的组成），用95％ 的 O 2和鼓泡取代脑脊液5％的CO 2）为高碳酸血症。申请的刺激30分钟（ 图2B）。 </li><li> 离体 ，诱导期后，在供以后使用（ 图3）冷冻保护溶液中2.5毫升4％PFA传送CNS新生小鼠或大鼠的一个2.5毫升管48小时，并储存于-18℃。 注意: 体外 ，这是没有必要灌注动物。先前的研究表明，小试样测量小于10毫米的直径可以通过简单的扩散22是固定的。渗透的引用率（距离，单位为mm，对于固定剂扩散到组织中），用于醛是2或3毫米/小时。 </l我&gt; </ol> 4.脑干切片注意:从该步骤到结束，该协议是严格用于体内和离体导入装置是相同的。 <ol><li>前切片，放置一个个体以及在一个12孔板插入并加入2.5倍毫升的PBS在每个孔中。 </li><li>使脑干的串行冠状切片（40微米）配有一个低温恒温器和收集它们在12孔板中。对于整个成年脑干，使约70切片（ 图3）。 注意:可以将部分的组织，从几个动物并联于一个板。 </li></ol> 5.免疫组织学程序（表1） 注意:在整个过程中，部分保留在井插入。 <ol><li> 1天-内源性过氧化物酶活破坏，非特异性结合位点的封锁，并与第一抗体孵育（图4） <ol><li>洗在室温10分钟，用0.​​1M PBS中的部分（2.5毫升，每孔）。重复3次。 </li><li>通过在RT与过氧化氢​​ħ孵育30分钟冠状切片抑制内源性过氧化物酶活2 O 2，3％在0.1M的PBS（2.5毫升，每孔）。 </li><li>洗部分在室温10分钟，用0.​​1M的PBS（2.5毫升，每孔）。重复3次。 </li><li>通过在RT用在PBST中的山羊血清（2％）温育1小时的冠状切片阻断非特异性结合位点。 </li><li> （:4000，SC-52，1）在PBST用牛血清白蛋白（0.25％）（2.5毫升，每孔）与抗C-FOS蛋白质的兔多克隆抗体孵育48小时的冠状切片在4℃下。 </li></ol></li><li> 第3天-孵育二次抗体和显色反应的发展（图4） <ol><li>洗部分10分钟，用PBS 0.1M的在RT（2.5毫升，每孔）。重复3次。 </li><li>孵育在PBST用牛血清白蛋白（0.25％）（2.5毫升，每孔）:在RT 1小时用生物素化的山羊抗兔抗体（500 1）冠状切片。 注意:研究者还可以使用耦合至荧光探针的二级抗体在这个阶段。在这种情况下，停止该协议在这个阶段，并用荧光显微镜直接检查。 </li><li>洗部分在室温10分钟用PBST（2.5毫升，每孔）。重复3次。 </li><li>孵育1小时的冠状切片与抗生物素蛋白 - 生物素 - 过氧化物酶复合物（1:250）在PBST（2.5毫升，每孔）。 </li><li>洗部分在室温10分钟用PBST（2.5毫升，每孔）。重复2次。 </li><li>洗部分在室温10分钟用0.05M Tris-缓冲液（2.5毫升，每孔）。重复2次。 </li><li>孵育0.02％3.3-二氨基联苯胺四盐酸盐，硫酸镍铵0.04％和0.01％的过氧化氢的冠状切片在0.05M Tris-缓冲液（pH7.6）中一个ŧRT（每孔2.0ml）中。在剩余溶液（25毫升）中添加17微升的 H 2 O 2（H 2 O 2溶液H中2 O 30％）（每孔2.0ml）中。 注:研究者应确定在光学显微镜下开发时间。然而，5至10分钟提供了良好的染色强度。 </li><li>当染色强度是最佳的（在光学显微镜下的控制，参见图5和图6），通过洗涤段在室温10分钟，用0.1M的PBS（2.5毫升，每孔）使反应停止。重复4次。然后，冲洗部分用蒸馏水。 </li></ol></li><li> 采样安装（处理与下化学罩护理和手套） <ol><li>山路段上连续幻灯片和风干。对于这一点，仔细涂抹几节rostro - 尾以便与幻灯片上的刷子。根据样品清楚标示的幻灯片。 </li><li>以绝对的人脱水cohol:浸入幻灯片在无水乙醇浴中在室温持续30秒。重复两次。 </li><li>清除用二甲苯:浸泡在二甲苯浴中的载玻片在室温3分钟。重复两次。 </li><li>盖玻片封固剂的幻灯片。对于这一点，应用5滴上滑动安装介质中，然后通过驱动出气泡申请玻璃盖。气干48小时。 </li></ol></li></ol> 6.数据和统计分析<ol><li>检查光镜下部分。目测计数，使用标准的地标23,24高倍率（200X）FLI神经元。在C-Fos的点状染色神经元细胞核本地化。 </li><li>对于每一个分析区域，计数每部分的c-FOS阳性细胞的数目和比较控制和刺激条件之间的平均数。根据数据的正常性，可以使用非配对学生t检验或Mann-Whitney检验。差异被认为若P显著0; 0.05。积FLI神经元上的图（放大100倍），使用的图管附连到显微镜用数码相机拍摄的（ 图5）的分布。 </li></ol>

<ol>
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The authors have nothing to disclose.

c-fos是一种立即早期基因，其产物的检测，所述c-fos蛋白，是古典用于识别体内 11,13,25,28参与具体呼吸道反应神经元群体和体外 16-18 27,32,33。 关键步骤的协议中 是在灌注一步小心。在4％PFA溶液必须很好制备和固定和后固定步骤必须足够长，以获得最佳的切片和染色。此外，启示是该过程的?...

所述C- fos基因在1980年1,2开始被确定为在第一时间和它的产物的特征在于在1984年为具有基因活化剂性能3,4-核蛋白质。它参与与神经刺激有关的长期机制。事实上，在神经元活动的变化导致第二信使信号级联诱导立即早期基因c-fos的表达，诱导产生的转录因子c-fos的。后者启动晚期基因的表达，从而参与神经系统的许多不同类型的刺激4适应性反应。因此，由于1980 5,6-结束时，C-FOS蛋白质检测已被经常用于研究的一般4和对中枢神经系统（CNS）的活性的外源因子对基因转录的影响为映射出神经通路参与不同的生理人的条件。 基础c-fos表达进行了研究不同物种包括老鼠，老鼠，猫，猴和人4。从而，其表达的动力学是相对众所周知的。转录激活是快速（5至20分钟）7,8，和mRNA的累积刺激9在发病后30和45分钟之间达到最大值并用12分钟的短半衰期下降。在c-fos蛋白合成如下mRNA的积累，可以通过免疫组化在20至90分钟后刺激6进行检测。 c-fos表达的分析在体内研究是经典用于鉴定参与低氧或高二氧化碳10-14的通气反应中枢性呼吸网络。最近，这个工具也用在体外脑干筹备探索呼吸中枢网络适应缺氧或Hypercapnia 15-18。事实上，这些制剂产生经典同化到呼吸中枢驱动器19有节奏的活动。因此，这种类型的制剂的具有被完全deafferented的优点，因此，对于c-fos表达的结果只反映中央的刺激所造成的后果，而不外 ​​围结构的任何干预。 在C-FOS检测可以通过免疫组化或immunohistofluorescence方法进行。间接免疫检测需要使用抗c-FOS一个初级抗体和针对在其中初级抗体产生的物质的第二抗体。对于免疫组织化学方法，所述二级抗体缀合与作用的基板（H 2 O 2为过氧化物酶）上的酶（过氧化物酶，例如）。酶反应的产物是由一个发色团（3.3二氨基联苯胺tetrahydrochlorid开发E），该污渍，并可以在光学显微镜下进行观察。该反应可以使用硫酸镍铵来增强。这些方法允许活性的神经元在不同生理挑战的检测，因此，鉴定和/或涉及的连续的生理反应外周和中枢途径的映射。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture plate 12-Well</td>
			<td style="width:71px;">Costa</td>
			<td style="width:119px;">35/3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:107px;width:216px;">15 mm Netwell inserts with mesh polyester membrane</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">3477</td>
			<td style="width:167px;">The 15mm diameter well inserts have 74&#181;m polyester mesh bottoms attached to polystyrene inserts</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">primary antibody (rabbit polyclonal antibody against the c-Fos protein)</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-52</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vectastain Elite ABC KIT&#160;</td>
			<td rowspan="2" style="width:71px;">Vector laboratories</td>
			<td rowspan="2" style="width:119px;">PK-6101</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">(Rabbit IgG-secondary antibody)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaH2PO4*2H2O</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">71505</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7907</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH 0.1N</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">43617</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyvinyl-Pyrrolidone</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PVP-360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7903</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene-glycol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">33068</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X100</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trisma HCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T5941</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trisma Base</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.3-diaminobenzidine tetrahydrochloride&#160;</td>
			<td style="width:71px;">Rockland</td>
			<td style="width:119px;">DAB50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nickel ammonium sulphate</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">12519</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">H2O2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">H1009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">33817</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Entellan Neo</td>
			<td style="width:71px;">Merck Millipore</td>
			<td style="width:119px;">107961</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slide&#160;</td>
			<td style="width:71px;">Thermo-scientific</td>
			<td style="width:119px;">1014356190</td>
			<td style="width:167px;">Superfrost ultraplus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover glass</td>
			<td style="width:71px;">Thermo-scientific</td>
			<td style="width:119px;">Q10143263NR1</td>
			<td style="width:167px;">24 x 60mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A2153</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

the c-fos protein immunohistological detection: a useful tool as a marker of central pathways involved in specific physiological responses in vivo and ex vivo

许多研究努力查明并参与特定的生理法规的大脑区域。的原癌基因c-fos的 ，立即早期基因，是响应于各种刺激表达在神经元。蛋白质产物可与免疫组织化学技术导致使用的c-FOS检测到映射显示其活性的改变的神经元群体可容易地检测到。在这篇文章中，我们将重点放在参与通气适应低氧高碳酸血症或脑干神经元群体的鉴定。两种方法进行了描述，以确定参与神经元群体在体内在动物和体外在deafferented脑干的制剂。 在体内 ，动物暴露于高碳酸血症或缺氧的气体混合物。 体外 ，deafferented制剂灌流缺氧或高二氧化碳人工脑脊液。在这两种情况下，无论是控制在体内动物或电子邮件点¯x体内制剂常氧和碳酸血症的条件下保持。这两种方法的比较允许神经元激活即原点，测定外周和/或中枢， 在体内和体外 ，脑干，收集，固定，并切成段。一旦切片制备的C-FOS蛋白质的免疫组化检测，以便确定由低氧或高二氧化碳刺激活化细胞的脑干组作出。标记的细胞在脑干呼吸结构进行计数。相比于控制条件下，低氧或高二氧化碳增加在那些从而构参与中枢性呼吸驱动的适应的神经通路的几个具体脑干部位的c-FOS标记的细胞的数目。

的C-FOS检测是一个有用的工具，它允许如在体内 （ 图2A）低氧高二氧化碳或模仿这些条件下离体 （ 图2B）的情况下在特定条件下活化细胞的识别基团。 在体内 ，新生儿，青少年或成人啮齿动物被放置在气密箱中的气体环境是由一气体混合物为30至180分钟，13,25,26（ 图2A）精确限定的组合物连续地更新。随?...

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The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

在c-fos蛋白免疫组织学检测:一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

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Research

JoVE Journal

Biology

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

33.2K 次观看.  University Paris 13. Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

视频: The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

科研

生物学

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 ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

在体内人细胞 Rb 蛋白 sumo 的检测与分析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

在c-fos蛋白免疫组织学检测:一个有用​​的工具，中央通路在特定的生理反应所涉及的标记

在c-fos蛋白免疫组织学检测:一个有用的工具，中央通路在特定的生理反应所涉及的标记