经费是由美国国家科学基金会CCLI＃0633618和俄亥俄州大学提供。某些菌株是由线虫遗传学中心提供。研究生Reetobrata八宿出现在视频中。

注：多协议的步骤都带有用于设置和准备根据实验的具体情况选择。对于这些情况，其他的步骤都为A，B，C 等有替代措施的协议在图1中概述。 1。聚赖氨酸包被的载玻片的制备三种不同类型的幻灯片可以被使用，这取决于制备的容易性，相对的粘合性，以及抗体的非特异性结合之间的期望折衷。的滑动制剂替代细节在下面的步骤1A-1C所示，为了提高密合性和复杂性的描述。制备这些不同的方法的幻灯片可以被一起用于单次实验。 1A。没有幻灯片准备市售的多聚赖氨酸包被的载玻片提供低粘附性和低背景。 1B。幻灯公关eparation最适合甲醇 - 丙酮固定中等的附着力和低背景。 <ol><li>制备聚赖氨酸溶液用于浸渍载玻片放入400毫克分子量非常高的（150,000 - 300,000 D）的聚-L-赖氨酸〜200毫升双蒸水。加入2ml 10％的叠氮化钠的股票（终浓度为0.1％）作为防腐剂。注意：干叠氮化钠是被动的和一切形式都是有毒的。戴上口罩和手套，而称重粉末，使10％的股票在双蒸水。 </li><li>擦拭单头磨砂载玻片用无绒布擦拭，去除污迹和颗粒。这应该甚至与指定购买的幻灯片进行“预清洗”。 </li><li>地方滑回至后端的幻灯片固定器，染色壶或其他片染色容器中，保持磨砂边缘并不太完全一致（使后来的分离更容易）。放置片支架中的烧杯中，用70％乙醇（无需试剂级醇），用1％HCl的二平息水或填充染色缸到结霜的水平。在最少5分钟解决泥石流。注意：此解决方案具有腐蚀性，戴手套。注：此溶液可以重复使用几次（长达数月）。 </li><li>在1分钟运行蒸馏水冲洗幻灯片。 </li><li>干燥载玻片完全在无尘环境中或者通过放置在40-60℃的烘箱中30-60分钟，或通过保持在室温下过夜。 </li><li>在摇臂15分钟孵育幻灯片聚赖氨酸溶液（包括unfrosted部分）。 </li><li>重复幻灯片的干燥。 </li><li>重复多聚赖氨酸溶液培养和干燥步骤。 </li><li>分离用细长的金属物体，如薄称重抹刀的幻灯片。如果它们正确地附着，它们很难分开。穿戴适当的安全装备（护目镜）和台式纸（使用后丢弃）工作，因为玻璃的小位可飞松动时，幻灯片是分开的。 </li><li>在干净的玻片储存（无尘）滑动箱，在4℃，直到需要。 </li></ol> 1C。玻片制备的最佳甲醛固定高粘着性，但高背景。 <ol><li>制备方法1B平放于无尘，烤箱安全菜（用于干燥烘箱），或在平坦的无尘表面（空气干燥）的地方幻灯片。 </li><li>放置在每个滑动中间的聚赖氨酸溶液的20-60微升下降。 </li><li>干燥载玻片完全在烘箱或在室温下进行。聚赖氨酸就会落后苍白椭圆离开，因为它蒸发。这些椭圆形的粘性非常高。不幸的是，它们也结合到第一和第二抗体，甚至阻断之后。 </li><li>在清洁（无尘）在4℃幻灯片格子铺的幻灯片，直到需要。 </li></ol> 2。冷冻线虫幻灯片的制备通过完全漂洗免费使用方法2A细菌（用于线虫板）或准备线虫进行染色2B（个别线虫）。 2A。从线虫板准备幻灯片<ol><li>放置在干冰平坦的金属片以预冷。 </li><li>用蒸馏水和玻璃吸管（减少病虫损失）或微量冲洗从NGM板蠕虫与细菌到1.5 ml离心管中。板可以同步或混合年龄，不要使用板与许多dauers（难以破解）或饿死蠕虫（增加自发荧光），除非必要的。 </li><li>冲洗蠕虫3-4倍，用蒸馏水冲洗直至无细菌。 （为了减少病虫损失使用相同的玻璃吸管。）沉淀蠕虫，要么让他们坐在板凳上3-5分钟（收集主要是大人管的底部）或旋转30秒〜1000转（500〜 G）在台式离心机（收集成人，幼虫和胚胎）。 </li><li>蠕虫除去大部分的水从管中，留下的水约两倍体积的蠕虫（但至少25μl总体积）。 </li><li&gt;“底”（在步骤1中处理）的标签磨砂边载玻片多聚赖氨酸与反不可磨灭的墨水和地点的幻灯片旁边的容器用干冰，标签的一面朝上。 </li><li>有相同数量的常规（擦拭干净，但不能多聚赖氨酸处理）“顶”可用幻灯片。 </li><li>地方〜在水25μl的蠕虫在“底部”粘附滑动并涂布到含有蠕虫比使用移液管尖端的侧面滑动的中央部的液体中。 </li><li>让蠕虫沉降30秒至2分钟。如果幻灯片是适当的粘性，该蠕虫的两端会坚持，因为他们接触的幻灯片。 </li><li>按住底部滑动，另一只手放在顶部滑过底部滑动，这样所有，但磨砂的部分是重叠的。液体应保持幻灯片略微分开，以使蠕虫仍然稍微移动，不要让幻灯片滑过彼此-这曲折的蠕虫 。 </li><li> 小心地按压直在S轻轻在上面滑动，用每只手的拇指和食指。随着压力的理想值，在幻灯片上边缘的最大的雌雄同体的有几个会破裂，而大多数的成虫和幼虫会联系每一张幻灯片，但将保持不变。 </li><li>立即小心地（ 不打滑）把幻灯片上的金属片上的干冰。幻灯片应该会出现一分钟内冻结。保持在干冰上5分钟，直到完全冻结。 </li><li>在这一点上，幻灯片可以被存储在在-80℃下的标记的框数天。 （如果他们是在-80℃下保持几个星期，水会开始升华，距离蠕虫不会弄脏以及）。如果幻灯片保存在-80℃，让他们开裂前干冰上10分钟“热身”。 </li><li>继续执行步骤3（固定）。 </li></ol> 2B。个人线虫幻灯片的制备<ol><li>放置一个扁平金属片上干冰PREC小山。 </li><li>使用蠕虫挑个别线虫转移到水对一个NGM板下降到细菌释放他们。线虫（S）应该是精心喂养，以减少自发荧光，如果可能的话。根据需要重复转移到新的水斑，直到蠕虫（s）是免费的细菌。 </li><li>标签的“底”聚赖氨酸玻片上柜台不可磨灭的墨水和地点的幻灯片磨砂侧旁容器用干冰，标签的一面朝上。具有相等数目的贴壁少“顶部”（未处理的）可滑动。 </li><li>将水5-10微升滴在底部滑动的双处理与聚赖氨酸的区域。如果传递更多蠕虫使用较大的音量，以防止水从完成前蒸发。 </li><li>蠕虫（次）转移到水滴与蠕虫挑，用显微镜观看的蠕虫下沉触摸粘滑动面。少到一个蠕虫传输到每下降多达20 +蠕虫。 </li><li>按住底部s立德，一手将上方滑过底部滑动，这样所有，但磨砂的部分是重叠的。 </li><li>立即小心地（ 不会滑倒或压缩 ）把幻灯片上的金属片上的干冰。保持在干冰上5-30分钟。 （不要将这些幻灯片长期来看，随着幻灯片容易干。） </li><li>继续执行步骤3（固定）。 </li></ol> 3。固定术固定剂是必要的&#39;修复&#39;的抗原发生在细胞，无论是沉淀（“轻固定”）或交联（“硬固”）的抗原。固定可能会破坏抗原性，大多数已知的抗体工作最好与一个特定的固定条件。对于新抗体，一系列的固定条件下进行测试。 对于任何固定术，第一步是使磷酸盐缓冲生理盐水溶液。下一步修复幻灯片使用方法的（光线虫抗体染色固定用甲醇和丙酮）或B（用甲醛或戊二醛更难修正）。 <ol><li>通过加入80 g氯化钠2.0克氯化钾27.2克Na 2 HPO 4•水平制备无菌的10×PBS（磷酸盐缓冲盐水）贮备液2 0，2.4克KH 2 PO 4，将20ml 10％的叠氮化钠的股票。为了使这种原料溶液，称出叠氮化钠的粉末，使10％的溶液在双蒸水中。生理盐水搅拌用文火直到溶解。注意：干叠氮化钠是被动的和一切形式都是有毒的。用干叠氮化钠或解决方案的工作时，应戴上口罩和手套。 </li><li>让盐水凉液（Tris pH值对温度比较敏感），然后pH值至7.2与1-10 M氢氧化钠。顶部至1 L双蒸水和高压灭菌消毒。在室温下储存，并稀释根据需要，用无菌双蒸水至1倍。注意：氢氧化钠是腐蚀性 - 在使用过程中戴手套。 </li></ol> 3A。用甲醇 - 丙酮光修正<ol><li>把100％甲醇，100％丙酮，和1x磷酸盐缓冲盐水（PBS）中的染色瓶在冰上预冷，至少10分钟。注意：甲醇和丙酮是有毒的，戴手套。 </li><li>取一对底部和顶部的幻灯片从干冰，迅速扭转他们分开，并立即浸泡在冰冷甲醇“底”幻灯片2分钟。 “顶”幻灯片可能被丢弃。 </li><li>传送载玻片从甲醇到冰冷的丙酮为4分钟。如果幻灯片有许多蠕虫，大多数（90％）将在修复脱落，即使有最好的准备幻灯片。如果单一的蠕虫切片准备妥当，蠕虫应保持粘合。 </li><li>放置或蘸用PBS（冲洗掉固定液）的jar幻灯片并继续执行步骤4（染色）。 </li></ol> 3B。使用甲醛或戊二醛固定硬<ol><li>稀释试剂级甲醛或戊二醛溶液在1x PBS至0.5-4％，终浓度。等份（1.5毫升）毫安Ÿ储存盖紧在-20至-30℃;请在使用前解冻冰等分。注意：甲醛溶液随着时间的推移，并与空气接触。注意：甲醛和戊二醛是有毒的，戴手套，在通风橱中工作。 </li><li>取一对底部和顶部的幻灯片从干冰，迅速扭转他们分开，并立即将“自下而上”的幻灯片与蠕虫在平盘盖。 （顶部滑动可以被丢弃）。 </li><li>立即吸取200微升有毒固定液在与蠕虫的幻灯片区域的中心。固定剂将代替部分冷冻，然后融化以覆盖滑动的更大区域。 </li><li>如果蠕虫不能完全覆盖固定液，用微量立即并小心地添加更多的固定液。不允许固定液流过滑动的边缘。 </li><li>盖上盖子的盘子和离开10分钟，以在室温下过夜。可以肯定的菜是加湿，如果再孵化ARe一起使用的 - 这可以通过增加折叠的湿无绒布擦拭物的菜来完成。不要让湿巾触摸幻灯片。 </li><li>固定完成后，认真吸取松散蠕虫固定液至1.5 ml管浸在一个染色缸，用PBS的幻灯片。 </li><li>旋管蠕虫，松了高速，持续30秒沉淀。用PBS冲洗蠕虫两次，然后在处理与幻灯片上的蠕虫（做在微量离心管中，而不是在幻灯片上步）平行。 </li><li>继续执行步骤4（染色）。 </li></ol> 4。抗体染色该抗体染色协议是类似的幻灯片上的任何组织标准协议。该协议是相同的所有幻灯片，无论准备步骤1-3。 <ol><li>制备抗体的缓冲液通过混合700毫升双蒸水，加入100毫升10×PBS中，加入5ml的Triton X-100（0.5％终浓度），将2​​ml的0.5M EDTA的pH为8（1 mM的终浓度），1克牛血清白蛋白（0.1％终浓度）， 5毫升10％的钠AZIDE溶液（0.05％最终）。 pH值至7.2，用HCl，然后双击加蒸馏水至1 L，在4℃下储存</li><li>准备块由重构驴血清用双蒸水，然后加入5毫升血清对45毫升抗体缓冲液（最终10％血清）。 </li><li>在抗体缓冲液加1％BSA稀释抗体准备原发性和继发抗体溶液。推荐稀释度是1:50-1:2,000初级抗体和1:1,000-1:5,000的二抗（Cy3or OG488标记的驴抗用于提高一抗的物种）。 </li><li>准备DAPI股票混合2.5 mg / ml的双蒸水，存放在冷冻在-30°C。8微升等份注意：DAPI是一种有毒的DNA结合染料，戴手套，而称重和分装成等分。 </li><li>准备10毫升安装中通过混合200毫克正丙基没食子酸酯，0.3毫升的1M Tris pH为9，2.7毫升15毫升锥形管中的双蒸水。热，在65℃下进行约10分钟，直至溶解。添加7毫升甘油和DAPI的8微升等分和涡好。分装培养基用1.5 ml离心管，包装箔，并且在黑暗储存在4℃（空气和光降解介质 - 如果它变得更黄，放弃在生化危机的废物）。注意：正丙酯是一种反应性的抗氧化剂，戴上手套时称重。 </li><li>幻灯片转移到一个染色缸带座1小时在室温或4℃过夜°C（最好过夜硬修复）。避免晃动，以避免失去蠕虫。 </li><li>转移到幻灯片染色缸与一抗或地方的幻灯片平板在湿润的菜和顶部各有100-500微升一抗。过夜孵育的幻灯片，在4°C，无晃动。 </li><li>经过初级抗体孵育，转移到幻灯片的PBS染色的罐子。 </li><li>滤波器模块通过漏斗衬有滤纸，并储存于4°C，如果无菌过滤，每1-2周，座可重复使用1个月或以上。同样，筛选和保存在4℃下再利用（多达10个或更多轮染色的）第一抗体。 </li><li>幻灯片冲洗三次（〜20每分钟）的抗体缓冲液。 </li><li>放置在第二抗体滑动不透光的染色容器孵育4小时，在室温下过夜或在4℃下</li><li>转移到幻灯片的PBS染色的罐子。过滤并保存第二抗体，在4℃下进行再利用（多达10个或更多轮的染色）。 </li><li>幻灯片冲洗三次（〜20每分钟）的抗体缓冲液。 </li><li>幻灯片转移PBS及PBS中离开（分钟到过夜，4℃），直到准备安装。 </li><li>迅速对个别幻灯片的工作，使幻灯片前面的蠕虫留湿。从PBS中删除一个幻灯片，干背幻灯片用无绒布擦拭，并放在纸巾。 </li><li>地方〜20微升的安装介质在蠕虫，以便有上滑动和倾斜滑道轻轻地混合使用这两种方案中，PBS大致相等的部分的。 CAUTION：安装介质是有毒的。 </li><li>倾斜滑动所以磨砂边缘较低，解集附近结霜，然后将上解24 x 60毫米盖玻片一边。 </li><li>轻轻放下盖玻片，以减少气泡（从而增加漂白和破坏观看）。如果气泡形成，提起盖玻片的边缘慢慢地，然后relower它， 而无需通过蠕虫滑动盖玻片 。 </li><li>仔细擦干幻灯片的边缘，而无需移动盖玻片，用不起毛的抹压缩的边缘。幻灯片的相对干燥的边缘必须是可见的所有周围的盖玻片的边缘</li><li>密封盖玻片用2-3层指甲油。幻灯片可以制备和贮存避光期间被放置在一个平面滑动支架。一旦滑动密封良好，干燥，清洁的幻灯片盖玻片和背部。 </li><li>密封良好的滑动，可以保持在黑暗中为天，在4℃或数周，在-20℃下一段时间后，DAPI站伊宁DNA和最绿色的染料（ 如 488 nm激发）褪色。使用任何油镜后，重新封装用盖玻片需要更多的指甲油， 如 。 </li></ol>

<ol>
	<li>Harlow, E., Lane, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibodies: A Laboratory Manual. , (1988).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Transformation.">DNA Transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Boulin, T., Etchberger, J. F., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene+fusions.">Reporter gene fusions.</a> WormBook. , (2006).</li><li>Duerr, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> WormBook. , (2006).</li><li>Finney, M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unc-86+gene+product+couples+cell+lineage+and+cell+identity+in+C.+elegans.">The unc-86 gene product couples cell lineage and cell identity in C. elegans.</a> Cell. 63, 895-905 (1990).</li><li>Garriga, G., Desai, C., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+control+the+direction+of+outgrowth,+branching+and+fasciculation+of+the+HSN+axons+of+Caenorhabditis+elegans.">Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans.</a> Development. 117, 1071-1087 (1993).</li><li>Miller, D. M., Shakes, D. C., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Caenorhabditis elegans: Modern Biological Analysis of an Organism. , 365-394 (1995).</li><li>Crittenden, S. L., Troemel, E. R., Evans, T. C., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLP-1+is+localized+to+the+mitotic+region+of+the+C.+elegans+germ+line.">GLP-1 is localized to the mitotic region of the C. elegans germ line.</a> Development. 120, 2901-2911 (1994).</li><li>Strome, S., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+visualization+of+germ-line-specific+cytoplasmic+granules+in+embryos,+larvae,+and+adults+of+Caenorhabditis+elegans.">Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 79, 1558-1562 (1982).</li><li>Albertson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+first+cleavage+spindle+in+nematode+embryos.">Formation of the first cleavage spindle in nematode embryos.</a> Dev. Biol. 101, 61-72 (1984).</li><li>Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using Antibodies: A Laboratory Manual. , (1999).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> WormBook. , (2006).</li></ol>

作者宣称，她已经没有竞争的财务权益。

冷冻裂化协议的几种方法进行抗体染色法在C 1 线虫，它提供了一个相对简单的方法来染色蠕虫，但需要特定的试剂和练习以获得最佳效果。关键步骤（ 图1所示 ）包括：1）玻片制备（见步骤1和线虫2）手动压缩（见步骤2和3）快速固定（参见步骤3）。首先，最大限度地线虫的幻灯片粘连，最好准备，而不是依靠商业准备的幻灯片幻灯片，高分子量（长聚合物），聚赖氨?...

来确定蛋白质的细胞和亚细胞定位，科学家们已经传统上标记的组织中选定的特异性识别特定蛋白1抗体。在一些模式生物（如C）线虫 ，抗体染色常常被替换为分子遗传学技术，它更迅速地取得成果。在这些转化的生物体与构建体包含启动子和感兴趣的和绿色荧光蛋白的基因的编码区之间的基因融合物。然而，分子技术受到了一些文物，其中包括问题知道真正的启动子和高拷贝数（ 线虫的常用技术）2,3的变化构建体的表达。因此，染色的抗体仍然是许多科学家研究体内蛋白质功能的目标。 染色组织中的抗体可以是困难的，因为一个tibodies可能仅识别在特定的构象1的抗原。例如，抗体可能只承认变性或完整抗原固定在一个特定的方式，可能无法识别抗原原位 。抗体染色的线虫的问题是由线虫的角质层形成一个相对不透水的屏障，阻断抗体的获得组织中的事实而加剧。 有用于抗体染色几种不同的方法在C。线虫 （在我们以前的工作检讨4）。染色完好“蠕虫”的方法被开发出来，冻结和解冻的相对硬的固定液（甲醛或戊二醛）的冻融帮助破解角质层允许的固定液5快速渗透。固定后，角质层中透化，以允许抗体的渗透;方法包括用还原剂，胶原酶，或两者5-7治疗。这些Treatments保存形态，但往往降低或破坏抗体识别。替代方法包括解剖8，允许抗体渗透。 “冷冻裂化”是获得对半完整蠕虫的内部，以允许抗体染色9-10的方法之一。冷冻裂解可以用各种定影条件下进行，同时避免胶原酶和还原处理。线虫放置2粘合剂片之间，冷冻，然后将载玻片分离，留下大部分线虫的底部滑动和许多在顶部滑动的角质层。底部的滑动可以放置在固定剂并用粘接线虫整个滑动是通过抗体染色程序转移。该方法确实存在两大困难。首先，它是难以适用的必要的压力分裂线虫，而不会严重变形他们正确的金额。其次，许多蠕虫将不发勾选要么幻灯片，将丢失的固定液或冲洗。然而，用适当的幻灯片和实践，该方法将迅速得到线虫与合理的形态，可与各种各样的固定剂和抗体的使用。 该方法可能会稍微改变，这取决于实验者的目标。如果单线虫的染色是理想的（ 例如 ，以确定单一的转化体是否已改变的抗体染色），然后以最大的粘着滑动（但更高的背景染色）都可以使用，如实验室制备的玻片用额外多聚赖氨酸（见下文）。对于甲醛或戊二醛固定，高附着力的幻灯片（实验室制备的聚赖氨酸玻片）应该使用，因为这些线虫的注视后坚持更差聚赖氨酸。如果野生型线虫正在用甲醇固定和/或丙酮，然后以较低的粘着滑动和较低的背景染色，应使用（商业或升aboratory准备的幻灯片）。如果多种抗体和固定剂被在实验室中使用，一个选择的幻灯片可以提前准备的时间和存储，直到需要。 访问线虫组织已经获得了后，抗体染色程序遵循标准方法（较长的孵化组织的特征，而不是细胞1,11）。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine slide (ESCO brand)</td>
			<td>Fisher</td>
			<td>12-545-78</td>
			<td>Sigma brand slides also suitable</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P1524</td>
			<td>IMPORTANT: Brand and high molecular weight of polylysine critical</td>
		 </tr>
		 <tr>
			<td>single frosted edge slides</td>
			<td>Fisher</td>
			<td>48312-002</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S2002-25G</td>
			<td>TOXIC, wear gloves and mask while weighing out powder to make 10% stock. Wear gloves when pipeting stock solution</td>
		 </tr>
		 <tr>
			<td>coplin staining jar</td>
			<td>VWR</td>
			<td>47751-792</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>5-slide mailer</td>
			<td>Electron Microscopy Science</td>
			<td>71549-01</td>
			<td>One of many different brands of small slide holder, suitable for staining</td>
		 </tr>
		 <tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>MK262159</td>
			<td>IMPORTANT: Regular formalin solutions (37% formaldehyde) are NOT suitable. Either 1) Dissolve crysatlaline paraformaldehyde in phosphate buffer and store in small aliquots frozen. Or 2) buy electron microscopy grade paraformaldehyde in aliquots. TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Glutaraldehyde solution, 25% in water</td>
			<td>Sigma</td>
			<td>G 5882</td>
			<td>IMPORTANT: purchase electron microscopy grade glutaraldehyde in ampules; TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>EM-9410</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>ICN820451</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Donkey Serum, lyophilized</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Cy3 Donkey anti-Mouse IgG multi-labeling</td>
			<td>Jackson Immunoresearch</td>
			<td>715-165-150</td>
			<td>This company is recommended for high quality multi-label (affinity depleted) antibodies. Select appropriate dye and antigen (animal used to raise primary antibody). </td>
		 </tr>
		 <tr>
			<td>n-propyl gallate</td>
			<td>Fisher</td>
			<td>ICN10274750</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride</td>
			<td>Sigma</td>
			<td>D 9542</td>
			<td>TOXIC. Wear gloves and dispense into small aliquots and feeze to minimize exposure</td>
		 </tr>
		 <tr>
			<td>Whatman #1 15 cm diameter filters</td>
			<td>Fisher</td>
			<td>09805G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip #1 1/2 rectangle 24 x 60 mm</td>
			<td>VWR</td>
			<td>48393-252</td>
			<td>#1 1/2 thickness is optically best</td>
		 </tr>
		 <tr>
			<td>Microscope slide folder</td>
			<td>VWR</td>
			<td>48429-092</td>
			<td>Other brands are suitable</td>
		 </tr>
		</tbody>
 </table>

antibody staining in c. elegans using &quot;freeze-cracking&quot;

染色C。线虫与抗体的相对不透水的角质层必须通过化学或机械方法来绕过。 “冷冻破解”是指用于两个贴壁幻灯片之间压缩线虫，冻结他们，拉幻灯片除了身体拉角质层从线虫的方法。冷冻裂解提供了一种简单而快速的方式来访问该组织未经化学处理，并且可以与各种固定剂的使用。然而，它导致许多样品的损失和所需要的压缩机械地扭曲了样品。需要实践最大化具有良好的形态样品的回收率。冷冻裂解可以为特定的固定条件下，样品的回收率，或低非特异性染色进行优化，但不是对所有参数在一次。对于需要非常坚硬的固定条件和容忍需要化学通透角质层的化学处理，treatmen抗体完整的线虫在溶液中t可以是优选的。如果抗体需要一个打火机修复或如果最佳固定条件是未知的，冷冻裂解提供快速测定的抗体，可以得到特定的亚细胞和细胞定位信息对于所感兴趣的抗原的非常有用的方法。

当蠕虫被正确压缩，破裂，并轻轻固定，几乎整个蠕虫可以访问抗体染色（ 见图2）。原子核由DAPI染色所指示的位置的指示哪个蜗杆的零件都完好无损当蠕虫经受较硬的固定剂如甲醛，蠕虫的形态可能会变形（如看到的不自然的波状外形在图3A-D的肌肉）。另一个常见的​​问题是不均匀的固定或特定组织中的渗透。这样的一个例子示于图3E-H，其中的肌肉被?...

Watch this Scientific Journal Video about Antibody Staining in C. Elegans Using "Freeze-Cracking" at JoVE.com

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

“不许动开裂，”用于暴露线虫C.内组织的方法线虫抗体用于蛋白质定位，被证实。

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

antibody-staining-in-c-elegans-using-freeze-cracking

Research

JoVE Journal

Biology

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

22.5K 次观看.  Ohio University.  “不许动开裂，”用于暴露线虫C.内组织的方法线虫抗体用于蛋白质定位，被证实。 

视频: Antibody Staining in C. Elegans Using "Freeze-Cracking"

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

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

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...