作者感谢他们的资金来源，霍华德休斯医学研究所本科科学教育计划，以威廉和玛丽学院;授予赞助商:美国国立卫生研究院（MSS）;授权号:R15NS067566。他们也承认支持，从艺术和科学学院的威廉与玛丽，生物系和学院与动物护理援助。

伦理声明:本方法中进行从繁殖地在威廉和玛丽学院驯养斑胸草雀。所有程序遵循防止虐待动物协会的指引23，并批准由威廉和玛丽的OLAW（实验动物福利办公室）动物福利保障（＃A3713-01）的学院和有机构动物护理和使用委员会（IACUC）批准（＃2013-06 -02-8721-dacris）。 1，鸡蛋收集和孵化<ol><li>建立在14点10光斑胸草雀对:暗周期。提供食物，水，干草和巢箱自由采食 。注:日常保养和繁殖的斑胸草雀得到了很好的描述23。 </li><li>准备一个标准的小鸡孵化器与倾斜的货架上。注意:保持在37.5人工培养箱（+ / - 1）℃，80 - 95％的湿度，加入水至培养箱的，每天的底部。允许培养箱中平衡两天使用前。 <ol><li>选择小料杯（5×7.6厘米）至少2.5厘米深，以在孵化器保持蛋。线的底部和杯用纸巾的两个层的下边缘，使得杯被填充，同时还允许将鸡蛋容易地滚动。将这些杯子在孵化器的倾斜货架。注意:太多的填充能抑制滚动，这将阻止成功孵化因胚胎附着在外壳内部。 </li></ol></li><li>收集鸡蛋在两个小时后的光周期的开始。注:此为一个给定的孵育时间，由于父母的孵化最大限度地减少在发展阶段的差异。 <ol><li>拿起鸡蛋轻轻地用拇指和食指在尖端和基部沿鸡蛋的长度。使用一个沉闷，柔和4B石墨铅笔标记从巢收集奠定了日期和时间。注意:较软的铅笔4B降低了铅笔打破易碎蛋壳的风险。 </li><li> 1.3.2）将1 - 5标记的鸡蛋在每个C在孵化器，使卵可自由滚动，以防止胚胎粘附到外壳的内部。注意:在一个杯子放置超过5个鸡蛋可以防止个别鸡蛋滚动，降低胚胎存活率。 </li></ol></li></ol> 2，从鸡蛋去除胚胎<ol><li>为了准备清扫，收集下列材料:干净的手术刀，细尖镊子，和两个额外的细尖镊子。将10×10cm的称量纸的解剖范围，解剖提供一个清洁，不吸水表面的基础上。注:称量纸是至关重要的，因为它可以轻松操控蛋黄，是切割精致的膜用手术刀最好的表面。 </li><li>在50ml的聚丙烯管中制备磷酸盐缓冲盐水（1X PBS）中的等分试样，并获取至少三个转移移液管和一个小的废物桶。如果固定胚胎，准备4％多聚甲醛（PFA）等分注意:PFA蒸气是有毒的，一ðPFA涉及的所有步骤应在通风橱内进行，以尽量减少风险。如果Flash冷冻胚胎，获得液氮，并保持它附近的解剖范围。不育是不是一个问题，这个夹层协议，但要确保所有的材料都是干净的。 </li><li>从孵化器中取出蛋需要在斑胸草雀升级指南22中描述的理想阶段的时间点。注:36小时培养后，取出鸡蛋剖析6期胚胎（ 图4A），22和56小时培养后，取出鸡蛋剖析阶段12个胚胎（ 图3A）22。 </li><li>使用光纤照明灯，通过蛋沿其纵轴和光泽光拿着它照亮室内蜡烛鸡蛋。放置受光的末端的蛋后面并定位蛋黄和胚胎发育。 <ol><li>定位手术刀上的蛋黄相对的一侧，沿着切蛋从尖端到基地淡淡的压力。 </li></ol></li><li>通过仔细地施加压力，用拇指和食指的蛋的尖端和基部，或轻轻撬开蛋壳沿切割用钳子取出蛋的内容物。打开鸡蛋称量纸的正上方，以使蛋黄轻轻地铺开。 </li><li>检查蛋黄的白色区域交界处，这是作为一个淡淡的白环包围胚胎发育可见光和定向使用超等蛋黄尖镊子使胚胎位于市中心。 注意:如果没有观察到微弱的白色圆圈蛋黄的表面上，采取细镊子，轻轻滚动蛋黄结束，直到胚胎是在蛋黄上面。注:对于阶段1 - 9 月22日 ，胚胎磁盘小于6毫米，直径是作为一个淡淡的，稍微不透明盘蛋黄表面上可见的-参见图1以供参考。对于阶段10岁及以上22，血池允许应用改进的Visibility胚胎，但要小心，以避免穿刺卵黄囊，因为这使得定位胚胎困难。 </li></ol> 3，分离胚胎从胚外组织<ol><li>刺破蛋黄的边缘，以纾缓压力（ 图1B），使整个蛋黄直径旁边的胚胎盘的长度单一的削减。重复做这些对角线，直到含有胚胎的卵黄的部分已经成功分离（ 图1B）。注意:该步骤使蛋黄质量保持不动，而是围绕胚胎切割时，防止损坏胚胎结构蛋黄的表面上的压力降低，可以更精确。 </li><li>除去卵黄的边缘与一个移液管。注意:移除尽可能多的蛋黄越好，但留下少量使胚胎不附着到称量纸和撕裂的干燥表面上。 </li><li>由dispens洗胚胎盘ING的1X PBS在朝向胚胎的底部成45°角。将移液管的尖端紧邻，不高于胚胎磁盘。如果附加的蛋黄需要被去除，转移胚胎到培养皿之前分离任何蛋黄上的称量纸的手术刀。注意:此方法在称量纸的表面移除胚胎。 </li><li>用移液管沿着用1X PBS的最小体积，以一个小的，塑料培养皿转移胚胎。通过直接添加更多的1X PBS在培养皿中并滴的1X PBS附近，但不是上，胚胎洗胚胎。摇动培养皿洗和除去残留的蛋黄，倾斜的培养皿中，并除去废物的1X PBS中的移液管。 注意:当1X PBS中被移除时，胚胎通常不附着在陪替氏培养皿的底部，因为塑料表面是光滑的残余1X PBS中。然而，更多的1X PBS可如果胚胎附着在培养皿中添加。 如果固定吨他胚，按照步骤3.5和3.6。如果闪存冻结胚胎，跳到步骤立刻3.8。 </li><li>如果原位杂交的固定在胚胎为，紧接在添加4％PFA中于培养皿淹没的胚胎。直接滴入4％PFA到胚胎的顶部，以压平;这可以防止胚胎卷曲。修复胚胎在4％PFA在4℃12小时。 <ol><li>固定后，在脱水分级甲醇（甲醇）溶液中并储存于-20℃下在100％MeOH中的胚胎。 </li></ol></li><li>如果胚胎是阶段1之间- 8月22日 ，除去卵黄膜附着在胚胎。注意:该步骤可以在或固定后进行。胚胎未满期8月22日时不易观察，因为他们坚持卵黄膜，遮蔽关键结构， 如图2A所示。 <ol><li>把手延伸超出交界超等镊子的区域中的膜的边缘。 Caref来自ully翻转胚胎过多次洗去剩余卵黄颗粒和松开胚盘的卵黄膜的附着力。注意:请勿触摸胚胎的中心，因为这会损害胚胎结构。 </li><li>如果间隙不结的区域和卵黄膜之间出现，轻轻刮开结与超等区尖镊子从卵黄膜松开。握卵黄膜与极细尖镊子轻轻拉离胚胎。如果需要的话，轻轻握住胚胎盘的周缘处接合的区域拉胚离卵黄膜。 </li><li>丢弃在生物危害1废弃物（生物安全级别1）去除后卵黄膜。 </li></ol></li><li>在开展对青少年萌芽阶段的ISH检测，遵循的标准整装原位杂交协议进行鸡胚24，25，但考虑以下建议。 <ol&gt; <li>以减少损坏胚胎的ISH过程中，使用一个单一的5毫升玻璃小瓶中的带螺丝帽每个胚胎。仅填充小瓶用2 - 3毫升溶液，以确保将胚胎完全淹没。章动药瓶垂直放置5毫升小瓶成发泡胶架（或任何架子，将让小瓶安全），它固定在章动。注意:这种预防措施防止胚胎被撕裂，当它与所述小瓶的横章动期间该盖接触而可能发生。 </li><li>胚胎阶段0 - 6月22日 ，治疗用5微克/毫升蛋白酶K在1X PTW 5分钟室温。在1X的PTW 12 22与10微克/毫升蛋白酶K 10分钟，在37℃，以减少背景染色-治疗胚胎上演7。 </li><li>以产生足够的显色反应来检测在阶段1的基因表达水平低的- 10 22，孵育用10μg/ ml的探针浓度为12小时。对于年龄较大的阶段，孵育1＆＃956克/毫升浓度的探针在60℃下</li></ol></li><li>如果速冻胚胎，迅速补充2 - 3毫升的1X PBS的培养皿以下解剖和倾斜培养皿的胚胎从卵黄颗粒分离。去除液体，然后重复2 - 3次，以除去所有的蛋黄。用细尖镊子，转移胚胎到预先标记的微量离心管中，并在液氮中快速冷冻，在-80存储℃。前</li></ol> 4。的EdU细胞增殖试验。注册成立并在斑胸草雀胚胎检测埃杜。 <ol><li>使用光纤照明灯来定位胚胎或蛋黄蜡烛的蛋。 </li><li>相反的鸡蛋的一侧标记的蛋内的胚胎或蛋黄的位置，以确保胚胎未在注射过程中损坏。 </li><li>线60毫米的塑料菜造型粘土和模具它在一个碗的形状保持鸡蛋在所需方向。标记点应该是为导向，以允许插入使用显微操作显微注射针。注:粘土将在显微注射和整个步骤4.11孵化稳定鸡蛋。 </li><li>拉两个玻璃毛细管显微注射针。钝一针戳孔进入卵在显着的位置。通过与矿物油backloading它和将它插入到微量制备第二微注射针注射。 </li><li>设置的解决方案，每次注射释放至59.8 NL上的微量量。 </li><li>加载显微注射针的10mM储备溶液的EdU。 </li><li>创建与钝玻璃毛细管标记点在shell一个洞，小心不要打碎壳或跌落壳片放入鸡蛋腔。定向蛋，使得孔是在一个45°角，从而使注射针直接插入通过腔到蛋黄或胚胎。 </li><li>利用显微操作，将加载针约为1.0厘米的空腔。 </li><li>注入的EdU溶液所需量直接到发育中的胚胎或蛋黄。注:最多478 NL埃杜可以在不导致胚胎死亡被注入。 </li><li>立即包裹鸡蛋和粘土架用保鲜膜的多层覆盖蛋和菜，以防止孵化过程中胚胎的干燥。塑料磁带的边缘换到培养皿的底部，以防止塑料在孵化过程中散开。注:保鲜膜应只解剖前立即删除。 </li><li>容许新增殖细胞以纳入的EdU孵育卵在37℃，直至所需的阶段就达到了。注射后，不要返回蛋倾斜在孵化器的货架上。放置在平稳的表面上的塑料包裹的粘土内衬菜培养箱内。 </li><li>取下保鲜膜，解剖胚胎下面的上述协议。修复胚胎在4％PFA和孵化12小时，在4℃下，如上所述。固定后，5分钟，并将其存储在新鲜的100％乙醇洗涤用100％乙醇（EtOH中）在-20℃直到进一步分析。 </li></ol> 5。教育」，点击"反应协议都在玻璃小瓶中进行以下步骤。 <ol><li>补充水分的胚胎具有以下的连续5分钟清洗: </li><li>乙醇，75％乙醇和25％的无菌去离子蒸馏水（SDD）的水，50％乙醇和50％的SDD水，25％乙醇和75％1X的PTW，100％1X的PTW </li><li>在100％1X的PTW，每次10分钟洗3次。 </li><li>通过将1份10X储备缓冲液（10微升）的9份稀释反应缓冲液添加剂（套件组分F）sdd的水（90微升）。 </li><li>通过混合制备反应混合物在单独的试管中加入如下:875微升PBS洗涤，20微升的CuSO 4，5微升叠氮化物，加入100μl稀释的反应缓冲液添加剂。注意:反应混合物的体积可以缩小。该反应会工作，如果胚胎是在反应混合物中完全覆盖。临用前加入稀释后的反应缓冲液添加剂（步骤5.3）。所有后续步骤都在黑暗中进行。盖上铝箔胚胎从光保护。 </li><li>用铝箔覆盖的小瓶中，并垂直孵育它们在室温下两小时通过将它们放置在连接到一个章动器一发泡胶机架上。 </li><li>在新鲜的1X PBS，每次10分钟洗胚胎3次。 </li><li>培养在新鲜的4％PFA中过夜胚胎在4℃下</li><li>在4℃下对每个店铺和10分钟在新鲜的1X PBS中的1X PBS洗涤3次盖上铝箔胚胎，直到进一步的分析。 </li></ol>

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M., Gahr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+receptors+in+the+embryonic+zebra+finch+hindbrain+suggest+a+function+for+maternal+androgens+in+perihatchingsurvival.">Androgen receptors in the embryonic zebra finch hindbrain suggest a function for maternal androgens in perihatchingsurvival.</a> Journal of Comparative Neurology. 453, 57-70 (2002).</li><li>Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick;+a+great+model+system+becomes+even+greater.">The chick; a great model system becomes even greater.</a> Dev Cell. 8, 9-17 (2005).</li><li>Kuenzel, W. J., Medina, L., Csillag, A., Perkel, D. J., Reiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+subpallium:+new+insights+into+structural+and+functional+subdivisions+occupying+the+lateral+subpallial+wall+and+their+embryological+origins.">The avian subpallium: new insights into structural and functional subdivisions occupying the lateral subpallial wall and their embryological origins.</a> Brain Research. 1424, 67-101 (2011).</li><li>Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+chicken+embryology+to+the+understanding+of+vertebrate+limb+development.">The contribution of chicken embryology to the understanding of vertebrate limb development.</a> Mech Dev. 121, 1019-1029 (2004).</li><li>Vergara, M. N., Canto-Soler, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rediscovering+the+chick+embryo+as+a+model+to+study+retinal+development.">Rediscovering the chick embryo as a model to study retinal development.</a> Neural Development. 7, (2012).</li><li>Davey, M. G., Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+as+a+model+for+embryonic+development.">The chicken as a model for embryonic development.</a> Cytogenet Genome Research. 117, 231-239 (2007).</li><li>Murray, J. R., Varian-Ramos, C. W., Welch, Z. S., Saha, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryological+staging+of+the+zebra+finch,+Taeniopygia+guttata.">Embryological staging of the zebra finch, Taeniopygia guttata.</a> J Morphol. , (2013).</li><li>Hawkins, P., Morton, D. B., Cameron, D., Cuthill, I., Francis, R., Freire, R., Gosler, A., Healy, S., Hudson, A., Inglis, I., Jones, A., Kirkwood, J., Lawton, M., Monaghan, P., Sherwin, C., Townsend, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+birds:+refinements+in+husbandry+and+procedures.+Fifth+report+of+BVAAWF/FRAME+/RSPCA/UFAW+Joint+Working+Group+on+Refinement.">Laboratory birds: refinements in husbandry and procedures. Fifth report of BVAAWF/FRAME /RSPCA/UFAW Joint Working Group on Refinement.</a> Lab Anim. , (2001).</li><li>Pearse II, R. V., Esshaki, D., Tabin, C. J., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analysis+of+intra-+and+extraarticular+connective+tissue.">Genome-wide expression analysis of intra- and extraarticular connective tissue.</a> Journal of Orthopaedic Research. 27, 427-434 (2009).</li><li>Pizard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridization+and+detection+of+RNAs+in+vertebrate+embryos+and+isolated+organs.">Whole-mount in situ hybridization and detection of RNAs in vertebrate embryos and isolated organs.</a> Current Protocols in Molecular Biology. 66, (2004).</li><li>Warren, M., Puskarczyk, K., Chapman, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+proliferation+studies+using+EdUlabeling.">Chick embryo proliferation studies using EdUlabeling.</a> Developmental Dynamics. 238, 944-949 (2009).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+syntesis+in+vivo.">A chemical method for fast and sensitive detection of DNA syntesis in vivo.</a> Proceedings of the National Academy of Sciences U S A. 105, 2415-2420 (2008).</li><li>Hoogesteijn, A. L., DeVoogd, T. J., Quimby, F. W., De Caprio, T., Kollias, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+impairment+in+zebra+finches+(Taeinopygia+guttata).">Reproductive impairment in zebra finches (Taeinopygia guttata).</a> Environmental Toxicology and Chemistry. 24, 219-223 (2005).</li><li>Winter, V., Williams, T. D., Elliott, J. 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+three-generational+study+of+In+ovo+exposure+to+PBDE-99+in+the+zebra+finch.">A three-generational study of In ovo exposure to PBDE-99 in the zebra finch.</a> Environmental Toxicology and Chemistry. 32, 562-568 (2013).</li><li>Kitulagodage, M., Buttemer, W. A., Astheimer, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+effects+of+fipronil+on+avian+reproduction+and+development:+maternal+transfer+of+fipronil+to+eggs+in+zebra+finch+Taeinopygia+guttata+and+in+ovo+exposure+in+chickens+Gallus+domesticus.">Adverse effects of fipronil on avian reproduction and development: maternal transfer of fipronil to eggs in zebra finch Taeinopygia guttata and in ovo exposure in chickens Gallus domesticus.</a> Ecotoxicology. 20, 653-660 (2011).</li><li>Hallinger, K. K., Zabransky, D. J., Kazmer, K. A., Cristol, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birdsong+differs+between+mercury-polluted+and+reference+sites.">Birdsong differs between mercury-polluted and reference sites.</a> The Auk. 127, 156-161 (2010).</li><li>Chen, C. C., Wada, K., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioactive+in+situ+Hybridization+for+Detecting+Diverse+Gene+Expression+Patterns+in+Tissue.">Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue.</a> Journal of Visualized Experiments. , (2012).</li><li>Chen, C. C., Balaban, E., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+Avian+Brain+Chimeras+Reveal+That+Large+Brain+Size+Differences+Are+Influenced+by+Cell&#8211;Interdependent+Processes.">Interspecies Avian Brain Chimeras Reveal That Large Brain Size Differences Are Influenced by Cell&#8211;Interdependent Processes.</a> PLoS One. 7, (2012).</li><li>Chen, C. C., Winkler, C. M., Pfenning, A. R., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+the+developing+avian+telencephalon:+Regional+timing+and+brain+subdivision+continuities.">Molecular profiling of the developing avian telencephalon: Regional timing and brain subdivision continuities.</a> The Journal of Comparative Neurology. 521, 3666-3701 (2013).</li></ol>

作者什么都没有透露。

一个胚胎分期指南22和基因组注释的最新发展使斑胸草雀的理想模式生物发育研究。然而，斑胸草雀的胚胎，这在阶段1的范围从3到7毫米的小尺寸和脆弱性- 10月22日 ，可以使解剖困难11，14。定位和干净地从蛋黄表面除去胚胎可以是具有挑战性的。该协议提供足够的细节来轻松地执行的程序。该协议表明，通常不为人所知，但都是必要的关键步骤，以确保成功剥离。例如，有必要离开蛋黄的胚胎和称量纸排​​除附着的片材之间的小层。 胚胎的两个查明和消除是困难的。要解决识别蛋黄表面上的胚胎，闪耀光蛋黄的正上方后，有从蛋中删除，并期待在蛋黄在45°角，以找到胚胎。一旦胚胎的位置，切割称量纸上，注意不要撕破胚胎的卵黄。 8 22，用于更好的可视化结构的-如果进一步应用包括成像解剖差异， 原位杂交，或细胞增殖测定法，它以除去卵黄膜在第1阶段是重要的。如果除去初期蛋黄或卵黄膜时遇到困难，解决胚胎在4％PFA洗的PBS洗涤，减少胚胎脆弱性之前。由所述剥离时首先除去卵黄膜，结构是清楚可见的和完好的斑胸草雀胚胎原位杂交执行之后。 埃杜限制是超过478 NL导致胚胎致死的剂量卷管理。然而，广大的剂量范围允许varyin克级别的增殖细胞标记的。 在此套件中使用的"点击"反应的是铜（I）催化 - 炔 - 叠氮环加成（的Cu（I）AAC）。在这个特定的反应中，含炔胸苷类似物分子（EDU）由活跃分裂的细胞中。在的EdU的炔基的DNA的螺旋结构突出，并且通过暴露于缀合至绿色，荧光分子，其结合到游离炔基叠氮化物分子进行检测。绿色荧光显示新增殖细胞中的胚胎。生物正交的叠氮化物和炔基可以防止非特异性染色，因为这些活性物种是不天然存在于生物体。另外，因为DNA不需要为了进行变性发生的反应，还依赖DNA的分析，可以容易地进行27。 这种方法的固有局限性在于体积小片段ility斑胸草雀的胚胎。去除胚胎的卵黄膜阶段1 - 15 22能导致损坏胚胎的结构，如果不小心执行。然而，该协议简​​化了夹层法，允许研究者使用早期胚胎阶段，研究了以前没有被深入研究的结构异常和基因的表达。该协议开辟了道路过多的细胞，分子检测，这将允许调查，以确定成年表现型的发展起源。例如，将有可能审核基因表达牵连的声乐学习各种环境条件下或在开发28，29,30,31的最早阶段下列药物治疗。虽然本文没有证明，这种方法可能会允许其他程序，如放射性原位杂交技术对斑胸草雀的组织切片和电/ 在OVØ 手术 32，33,34。鉴于斑胸草雀已被确立为文学的一个巨大的身体内一个重要的模式生物，这些研究提供了尚未开发的机会连结发育机制与成人的生理和行为，语言7,9的特别发展。

这项技术的总体目标是从胚胎中广泛发育研究中的应用最早阶段得到斑胸草雀（Taeniopygia弄蝶 ）胚胎。斑胸草雀已成为主要的鸣禽模型有机体，并已被广泛用于各种领域，包括毒理学1,2，行为3，记忆和学习4,5,6，比较解剖学7,8，和语言发育9,10 。正如一个基因组测序的唯一女歌手，斑胸草雀允许雀形目顺序，代表已知的鸟类11，12,13 50％以上的分子和基因研究。 尽管字段的多样化使用成年和少年斑胸草雀的，一些研究已经在斑胸草雀的胚胎进行的，特别是在发展的早期阶段。这可以归因于它们的卵一个小尺寸ND胚胎，他们的新身份作为模式生物为14,15,16，其中鸡（ 鸡内金 ）以前被用来作为一个主要的模型系统17,18,19,20,21研究。然而，由于非声乐学习者，鸡不学习声乐学习，声乐学习，遗传，行为和参与运动学习10皮质基底节电路发展的遗传基础的适当的模型系统。 要注意的是斑胸草雀胚胎细腻得多和更容易被损坏比解剖和分子过程中鸡胚是很重要的。特别是，更大的关怀正在执行的斑胸草雀胚胎通透步骤时需要。强洗涤剂和酶，不会伤害一只小鸡胚胎会损伤斑胸草雀的胚胎。在一般护理方面，有必要把斑胸草雀卵产在小杯子放置在之前的孵化器以防止其滚动时，孵化过程中断裂。 斑胸草雀是服从行为研究，并容易滋生多产全年圈养，并且是声乐学习者。这些特性允许使用斑胸草雀的解决需要一个模型有机体，集开发，遗传学和语言行为方面。下文详述的解剖方法，结合具体斑胸草雀22最近开发的分期指南，使斑胸草雀的越来越有用规范化发展的模式生物。然而，获得的胚胎在早期阶段可以是艰巨的。该协议允许调查人员很容易地获得早期胚胎。调查研究复杂的行为在斑胸草雀，或发展其他小的毒理作用的早期发展和发育的分子基础，雀形目鸟类会发现这个解剖方法非常有用。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chicken egg incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">We use a Picture Window Hova-Bator Incubator, Circulated Air Model&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Click-iT EdU Alexa Fluor 488 Imaging kit</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Detection of cell proliferation.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope&#160;</td>
			<td></td>
			<td></td>
			<td>We use Olympus SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7&#39;&#39; Drummond capillary for Nanoject II injector</td>
			<td>Drummond</td>
			<td>3-00-203-G/X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drummond Nanoject II microinjector</td>
			<td colspan="2">Drummond</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Tweezers #55</td>
			<td>World Precision Instruments</td>
			<td align="right">14099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol (EtOH)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL Falcon tube (polypropylene)</td>
			<td>Fisher Scientific</td>
			<td>06-443-18&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep Lysis Matrix H tubes</td>
			<td>MP biomedicals</td>
			<td>6917-100</td>
			<td>Used for RNA extraction, used to homogenize embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber optic illuminator lamps (High Intensity Illuminator)</td>
			<td>Dolan-Jenner Industries&#160;</td>
			<td colspan="2">Fiber-Lite MI-150&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass vials with screw cap (DEPC treated)</td>
			<td>Fisher Scientific</td>
			<td>03-338A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge eppe tube (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M-8410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling Clay</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narshige PB-7 needle puller</td>
			<td>Narshige</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Omni Bead Ruptor Homogenizer</td>
			<td style="width:71px;">OMNI International</td>
			<td>19-010</td>
			<td>Used for RNA extraction</td>
		</tr>
		<tr height="25">
			<td colspan="3" height="25" style="height:25px;">4% Paraformaldehyde (4% PFA): 20 mL 8% PFA (32g paraformaldehyde, 350mL sdd H2O, adjust pH to 7.6, 400mL sdd H2O), 20mL 2xPBS</td>
			<td>Fixation of embryos. Caution, is harmful and do not inhale.&#160;</td>
		</tr>
		<tr height="25">
			<td colspan="4" height="25" style="height:25px;">Phosphate buffered saline (1xPBS): 200mL 10X PBS, 1800 mL Barnstead H2O, adjust pH to 7.4</td>
		</tr>
		<tr height="25">
			<td colspan="4" height="25" style="height:25px;">Phosphate buffered saline (10xPBS): 800mL Barnstead, 2.013g KCL, 80.063g NaCl, 2.722g KH2PO4 monobasic, 14.196g Na2HPO4 dibasic, 200mL Barnstead H2O, adjust pH to 6.5</td>
		</tr>
		<tr height="25">
			<td colspan="4" height="25" style="height:25px;">Phosphate buffered saline with 0.1% Tween-20 (1xPTw): 1800mL Barnstead H2O, 200mL 10X PBS, adjust pH to 7.4, 2mL Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mL Plastic petri dish&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic wrap</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prepease RNA Spin Kit</td>
			<td>PrepEase, Affymetrix</td>
			<td>78766 1 KT</td>
			<td>Used for RNA extraction, used to homogenize embryos</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Reaction Mix: 875&#956;L 1xPBS, 100mM 20&#956;L CuSO4 (Kit Component E), 5&#956;L Alexa Fluor 488 azide (Kit Component B), 100&#956;L diluted reaction buffer additive (Kit Component F)</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Detection of cell proliferation.&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">sdd H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seed cup&#160;</td>
			<td></td>
			<td></td>
			<td>We use it as a container when incubating eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel scalpel&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard nest box</td>
			<td></td>
			<td></td>
			<td>We use Abba Plastic Finch Nestboxes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4mL, Teflon Lined Cap, Glass Vials</td>
			<td>Fisher Scientific</td>
			<td>02-912-352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes (polyethylene)</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4x4 weigh paper</td>
			<td>Fisher Scientific</td>
			<td>09-898-12B</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and downstream analysis of zebra finch embryos at early stages of development

斑胸草雀（Taeniopygia弄蝶 ）已成为一个日益重要的模式生物在许多领域的研究，包括毒理学1，2，行为3，记忆和学习4,5,6。正如一个基因组测序的唯一女歌手，斑胸草雀具有在发育研究利用潜力巨大;然而，斑胸草雀发展的早期阶段，还没有得到很好的研究。在斑胸草雀的发展缺乏研究可以归结为解剖小蛋和胚胎的难度。下面的解剖方法，最大限度地减少胚胎组织损伤，这使得形态和基因表达在胚胎发育的各个阶段的调查。这就允许既明场和胚胎质量荧光成像，在分子的程序，例如，原位杂交（ISH），细胞增殖试验，并提取RNA进行定量测定如定量意图使用L-time PCR检测（qtRT-PCR）技术，这种技术可以让研究人员研究开发的早期阶段，这在以前是难以进入。

图解图1中的步骤表明胚胎的外观，同时附着在卵黄膜（A）和示范正确的方法，将胚胎从卵黄（B）区分开来。胚胎可以通过结点，它比卵黄膜轻得多的区域来识别。胚胎本身常常难以区分，直到蛋黄被切去。一旦胚胎从卵解剖，它可以是固定的或者快速冷冻以供将来使用。如果一个原位杂交计划在解剖胚胎，它必须除去，是通过结的带粘接到胚胎卵黄膜， 图2示出了胚胎的改进的可视性，一旦该膜被除去（C）的和适当的方式来剥离卵黄膜（A，B）。解剖和固定后，整体原位杂交进行观察， 如图3（A，A&#39;，B，B&#39;）和图4（A，A&#39;，B，B&#39;）和图5（A，B，C）检测orthodenticle同源2（OTX2）表达的胚胎发育暴露于低剂量的甲基汞的差异。 图5显示了感探头结果，证明缺乏背景。在图4中，尽管从蛋在同一时间点被解剖，发育受到甲基汞胚胎发展到第5阶段22（B，B&#39;），而对照胚胎发展到阶段6月22日 （A，A &#39;）。该组胚胎解剖和图4中所示从巢收集并从培养箱采取在同一时间。虽然一些天然变异存在于开发，基于先前的解剖数据，这是不可能的温度波动在孵化器将导致仅在2.4 ppm的甲基汞胚胎是开发板pmentally延迟。分阶段的差异表明在胚胎发育受到甲基汞改变细胞的增殖。 解剖前，埃杜注入每天2鸡蛋，并使其孵育过夜。解剖台16 22胚和固定后，埃杜是使用"点击"化学，允许检测增殖细胞如在图6（A，B，C），看到可视化。它放置在鸡蛋孵化器，并在解剖，为甲基汞暴露或执行埃杜法可能会破坏发育进程密切监测时间点是非常重要的。最早注射在第0天，这是收集的当天作为在步骤1.3中指定执行。这个胚胎解剖约38小时后（第一阶段7月22日 ）。存活率被发现是大约90％（同样的速度控制胚胎）只要在注入量为478下NL。 22胚后，一个RNA的提取是根据制造商的方案，没有必要进行优化， 如图7所示。卵黄膜的去除是不必要的用于RNA提取和后来的qRT-PCR应用。 注:所有胚胎的数字被定向，以使前部和后部区域是在图像的顶部和底部。 <img alt="图1" src="/files/ftp_upload/51596/51596fig1highres.jpg" /> 图1。程序定位和解剖斑胸草雀的胚胎，阶段1-10 22。轻轻滚动，直到蛋黄幽幽白盘明显（一）定位胚胎。一旦胚胎位于蛋黄的中心，蛋黄被解剖以逐步的方式（B）如首次降息缓解（1）及随后的切割（2）边境交界处（ZJ）的地带，是卵黄膜压力附着在卵黄膜。刻度条表示1毫米。 <img alt="图2" src="/files/ftp_upload/51596/51596fig2highres.jpg" /> 图2。去除卵黄膜和胚胎结构的可见性。在除去胚胎从卵黄，胚发生在含4％PFA培养皿。如果一个原位杂交需要被执行时，胚胎结构的可见度是必不可少的，并且可以通过除去卵黄膜（A）来实现。握卵黄膜与极细尖镊子，轻轻地撕掉它远离胚胎直接在最外边缘处理的胚胎，如果有必要（B）。卵黄膜去除增加清晰度胚胎结构，并且允许胚胎原位杂交（C）进行成像或处理用。标尺代表1毫米。 <a href="https://www.jove.com/files/ftp_upload/51596/51596fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51596/51596fig3highres.jpg" /> 图3。整装原位杂交对斑胸草雀胚胎发育受到 orthodenticle同源2（OTX2）的甲基汞。表达模式进行了表征暴露于0.0 ppm的甲基汞（A，A&#39;）和2.4 ppm的甲基汞（B，B的胚胎&#39;），通过父母的饮食。该做的RSAL（A）和OTX2腹（A&#39;）的表达是可见整个中脑和视神经囊泡中阶段12月22日 ，治疗组的胚胎进行了解剖在同一时间点，但被发展迟缓看到在背侧（B）和腹侧（B&#39;）视图头部结构，这是舞台11 22缩略语的特点:MB，脑;运算，视泡。刻度条表示1毫米。 <img alt="图4" src="/files/ftp_upload/51596/51596fig4highres.jpg" /> 图4。整装 &#160; 原位 &#160; 对斑胸草雀胚胎发育受到 orthodenticle同源2（OTX2）的甲基汞。表达模式杂交进行了表征在第一阶段6月22日安莉芳<html> OS暴露于0.0 ppm的甲基汞（A，A&#39;）和第5阶段22胚胎暴露于2.4 ppm的甲基汞（B，B&#39;通过父母的饮食）。缩写:上午，中胚层的前缘;没有，脊索，脊索中胚层;婆，proamnion，前胚; PS，原条22。刻度条表示1毫米。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51596/51596fig5highres.jpg" width="600" /> 图5。整体原位装载 &#160; 使用感探头对斑胸草雀的胚胎进行杂交（A）第5阶段胚胎22（B）早 ​​期6 22胎。（c）第三阶段11 22胚胎。刻度条表示1毫米。 6"SRC ="/ files/ftp_upload/51596/51596fig6highres.jpg"/&gt; 图6。注册成立的EdU和去TECTION在斑胸草雀的胚胎。教育」按「化学法检测增殖细胞中的一个阶段16 22胚（A，B，C）。的EdU掺入到DNA中胸腺嘧啶26,27的地方，并使用点击化学27被检测到。增殖是清晰可见的体节的横向边缘，而tailbud。图A显示了胚胎后发生的完全扩散，也显示出个人增殖细胞。面板B显示了在整个胚胎增殖位置。面板C显示了前部区域，并显示出高度增生端脑（TE）中更详细。简称:AF，羊水折; FLB，前肢芽; HLB，后肢芽;乐，晶状体囊;毫秒，脑;公吨，后脑; OPC，视杯;每年，咽弓; SM，体节中胚层; TB，tailbud;德，端脑。标尺代表1毫米。 <a href="https://www.jove.com/files/ftp_upload/51596/51596fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/51596/51596fig7highres.jpg" /> 图7。质量从解剖斑胸草雀的胚胎。控制（0.0 ppm）的1.2 ppm的甲基汞胚胎中提取的RNA进行了解剖，并按照步骤3.8中所述快速冷冻。每个通道显示从每个治疗组中的两个胚胎匀浆提取的RNA。

Watch this Scientific Journal Video about Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development at JoVE.com

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications.

解剖和斑马雀胚胎下游分析在发展的早期阶段

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and ...

dissection-downstream-analysis-zebra-finch-embryos-at-early-stages

Research

JoVE Journal

Biology

10.8K 次观看.  College of William and Mary. The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications. 

视频: Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

科研

生物学

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

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 ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

可视化吞噬的早期阶段

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...