Name: a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis
Uploaded: 2016-03-15T15:00:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

这项工作是通过DFG项目＃JA2108 / 2-1资金支持DJJ。

注:以下步骤概括我们的方法对L.的胚胎和幼体阶段进行的现场试验stagnalis。当一个步骤涉及使用危险化学品这是由词&#39;小心&#39;和所有适当的安全程序的表示应采用。在补充文件1提供链接代表MSDS表的危险化学品。所有试剂配方在补充文件2提供。 1.解封设备的组装<ol><li>要做到这一点，连接20ml的一次性注射器，以硅管（1毫米的内径为3毫米的外径），使用P1,000尖切至适当的长度， 如图2A所示。 </li><li>磁带标准显微镜幻灯片倒大培养皿。胶带紧邻显微镜载玻片的硅管， 如图60;图2C和2D。 </li><li>搁置在显微镜载玻片一个拉玻璃针，轻轻将其插入硅导管，直到针尖突出的横跨管件的内径的方式约20％（参见图2C和2D）。一旦针在定位磁带下来的培养皿。 </li><li>允许在硅管的自由端，以另一培养皿，将收集的解封装材料休息。 </li></ol> 2.样品收集，固定和解封注:所有步骤都在室温下进行，除非另有说明。 <ol><li>仔细收集来自水族馆的墙壁鸡蛋串。要做到这一点，使用一块平坦的柔性塑料作为铲刮鸡蛋串从基材，并用塑料滤茶鱼浮蛋串出水面。阶段，并在图提供了使用导在显微镜下材料排序1。 </li><li>鸡蛋串放置到纸巾，并沿着使用轻量级镊子的卵块的纵向切口。滚动蛋胶囊出来的蛋串的，并通过使用轻量级镊子推动他们周围的纸巾从每个胶囊除去尽可能多的果冻材料尽可能的。 </li><li>使用轻量级镊子，转移蛋胶囊成含有5毫升自来水的皮氏培养皿。继续收集所需的发育阶段的脱果冻卵胶囊进入这个菜。收集所有发育阶段足够胶囊计划WMISH实验然后进行下一个步骤。 </li><li>当与较发达的幼虫工作（后5天第一次卵裂（DPFC）及以上）前麻醉他们固定。 注意:这将防止肌肉染上这使得解释原位染色模式非常困难。 <ol><li>放松幼虫（而他们仍然在他们的capsu莱）中的MgCl 2•6H 2 O:用于固定前30分钟2％w / v的溶液。 </li><li>通过在固定剂溶液中的蛋胶囊仍然浸没几幼虫而和监测在显微镜下它们的响应评估30分钟后放松程度。不完全放松的幼虫会缩回到他们的炮弹，而完全放松幼虫会不会响应。一旦这些幼虫已放宽继续进行下一步。 </li></ol></li><li>转移使用宽孔塑料吸管蛋胶囊成可密封管提供蛋胶囊10倍的体积（ 如 ，将1ml定居胶囊将需要10+毫升管）。 </li><li>抽吸尽可能多的液体尽可能从管中并用固定剂溶液的体积是10倍的定居蛋胶囊的体积替换。在RT轻轻转动在固定剂蛋胶囊的适当的时间为每个发育阶段（参见图1）。 </li><li> DiscontinUE旋转并允许胶囊下沉，吸固定液到合适的废物容器中。 </li><li>通过用0.1％吐温-20（PBTw）替换用磷酸盐缓冲盐水固定液和旋转在RT 5分钟洗蛋胶囊。吸出PBTw，重复两次。 </li><li>除去胚胎和使用该装置的胶囊幼虫（详见第1部分）在图2中所示的向上绘制胶囊进20毫升注射器，通过管道并经过玻璃针伸到连接管，然后消除胶囊集合菜。 注意:通常情况下，所有的胶囊的大部分（&gt; 90％）应该只需要一个通过该装置通。在许多情况下，胶囊型膜被破坏，但胚胎和幼虫仍破裂胶囊内部。通过简单地画起来到注射器中，再消除它重新处理该材料。 </li><li>收集胚胎解封和幼虫到1.5毫升管使用麦克风ropipette（为0 P20 - 3天老幼虫，并为与尖端的端部较旧的幼虫P200的切断）。 注:暂停（长达数月）中的协议可以在这个阶段进行。如果需要的话，该decapsuled材料应被收集到1.5ml试管进行存储（继续到下一个步骤）。否则立即继续协议3。 </li><li>允许胚胎和幼虫下沉到管的底部，并吸出上清液。用33％乙醇（乙醇）在PBTw更换，让坐5 - 10分钟。 66％乙醇中PBTw和100％的乙醇重复。在RT在100％EtOH中洗幼体两次。在100％的乙醇-20℃保存材料。 </li><li>当准备继续，重新水合物通过去除100％的乙醇和PBTw 66％乙醇代替样本，让我们坐5 - 10分钟。用33％乙醇重复PBTw，让坐在5 - 10分钟。最后用PBTw的3倍5分钟洗洗澡以保证所有的乙醇被删除。 </li></ol> 3.蛋白酶K，TEA和Post-Fi无线xation 注:我们发现在执行篮子小下面的步骤与网格地板最有效和最温和的方法快速交换时间关键型解决方案。虽然这些可以自制，我们使用的是与Intavis InSituPro -Vsi液体处理机器人（www.intavis.de/products/automated-ish-and-ihc）兼容篮（中等大小）。这样筐可以快速并容易地以一个12孔组织培养皿（TCD）的孔之间移动交换的解决方案，或该溶液可从井使用移液管被吸出。另外，可以在没有这些篮子只需吸从幼虫上清执行的所有解决方案的交流。在这种情况下，轻轻旋转运动将集中所有​​的胚胎和幼虫的菜允许从井的边缘除去上清液的中心。下面假设用户采用的解决方案交流篮子。 <ol><li>用移液管，移植胚胎和幼虫成一篮子坐在12以及TCD用2毫升PBTw的。 注:可以添加依赖于发育阶段的胚胎和/或幼虫的数量正在调查，但一般的经验法则是保持无胚胎/幼虫（ 即建筑面积的25％以上，不过度拥挤的篮子）。 </li><li>用2ml合适的蛋白酶K溶液（参见图1）的，和另外2孔与每个2毫升0.2％甘氨酸制备的相邻井。移动每个篮子到蛋白酶-K的孔含有适当浓度，并立即开始计时。 </li><li> 10分钟后移至各筐成孔含有0.2％甘氨酸和孵育5分钟。然后每筐移动到第二井用0.2％甘氨酸和孵育5分钟。洗出甘氨酸用3ml PBTw的3倍5分钟的交流。 </li><li>取出PBTw溶液，并用新鲜配制的三乙醇胺（TEA）溶胶曾经治疗样本ution 5分钟小心！不要搅动。用3毫升5分钟新鲜制备的TEA溶液替换。不要搅动。吸多数来自样品TEA溶液。加入与乙酸酐（TEAAA）解决方案新鲜配制三乙醇胺和孵育5分钟。 注意！不要搅动。 </li><li>可选 - 虽然上述步骤孵化，准备新一轮的新一批TEAAA解决方案，并重复上述步骤。 注意:这与TEAAA第二次处理是可选的，但可能有助于完全消除所有的背景，探头容易产生背景。 </li><li>通过从井中吸取它取出TEAAA溶液并用3ml PBTw的替换。不要搅动。取出PBTw和应用3毫升3.7％甲醛PBTw。 30分钟温育过程中轻轻偶尔摇动。 </li><li>由井吸它取出固定液，并用3ml PBTw的更换。材料转移到1.5ml试管。 - [RE放置PBTw与杂交缓冲液，并在室温下孵育5分钟 - 注意。 </li><li>管放入在RT热块，并设置温度到所需的杂交温度。 注:杂交温度探测器专用，将需要凭经验优化，但是我们发现55°C是一个很好的温度开始审判。允许块来杂交温度，并允许样品预杂交约15分钟（ 即，它需要制备的核糖核酸探针，更长的时间是没有必要的）。准备足够的体积（通常在100微升）稀释核糖核酸探针的。我们的100和500 ng / ml的典型试验探针浓度为初始范围。我们按照12准备的核糖核酸探针。 </li><li>从样品中除去杂交缓冲液，并在杂交缓冲液中添加所述探针的样品。覆盖用100μl（或足够的量，以形成杂交buffe上方的相矿物油中的R）。 注:矿物油防止广泛凝聚，将在漫长的杂交步骤形成。广泛缩合将显著改变杂交溶液两者的化学和探针的浓度。 </li><li>变性探针和样品加热至75℃10分钟，靶RNA，然后加热减少到所需的杂交温度。允许杂交进行了至少12小时（O / N）或更长的时间（24 - 48小时）。 </li><li>在杂交从热块删除单个管，拇指和食指之间迅速将其旋转到暂停幼虫，而不会干扰油相，并在加热块代替它。重复此每隔6次 - 12小时左右。 </li></ol> 4.热清洗和免疫检测注意:尽管我们使用液体处理机器人为执行以下步骤，这些也可以很容易地手动完成。在这种情况下，胚胎和幼虫抄ULD被保持在它们在杂交1.5毫升管。随后的所有溶液交换吸出，并用P1,000吸管加入。当手动进行的各步骤如下应该使用各1ml溶液。 <ol><li>热充足体积（每一个用于每个样品3ml）中的4倍，2倍和1倍洗涤溶液的杂交温度。 </li><li>洗所有的样品在4×洗涤缓冲液三次，每次15分钟，在杂交温度。洗所有的样品在2×洗涤缓冲液三次，每次15分钟，在杂交温度。洗所有样品在1×洗涤缓冲液三次，每次15分钟，在杂交温度。 </li><li>与在杂交温度1×氯化钠柠檬酸钠缓冲液+ 0.1％吐温（SSC + 0.1％吐温）洗涤一次所有样本。允许样品冷却到室温。 </li><li>清洗所有样本在两次1X SSC + 0.1％吐温15分钟替换用马来酸缓冲器（MAB）本1X SSC的解决方案，并让坐在10分钟重复MABW¯¯灰。用块替换解决方案MAB孵育1.5小时。 </li><li>换来的抗体溶液块溶液（1:10,000稀释在封闭液抗体），并在室温下孵育12小时（O / N），轻轻摇动。 </li></ol> 5.颜色开发与安装<ol><li>吸出抗体溶液并用PBTw洗15次，每次10分钟。替换PBTw用1×碱性磷酸酶缓冲液（AP）和孵育10分钟。 </li><li>虽然上述培养步骤的进行，准备AP染色溶液（见补充文件2） -注意。材料转移到TCD井和更换AP染色溶液1倍AP解决方案。注意现在时间，使得时间长度显色反应发生为可以被记录。监测染色模式的发展，直到信号背景比是最佳的。 </li><li>要停止显色，去掉1个AP染色溶液（配置在适当的赤身Ë容器），并申请2毫升PBTw，现在记下时间。冲洗PBTw两次，每次5分钟。使用这些漂洗之一将材料转移到1.5ml试管。 </li><li>取出PBTw和PBTw适用1毫升3.7％的甲醛和搅动或在室温为旋转至少30分钟（这也可以做O / N）。 </li><li>洗出固定剂与PBT的洗涤3次（配置适当的废物容器中的废定影液）。洗样品3次，在50℃下在去离子水中。 注意:这些洗涤消除盐的沉淀清除和可视化步骤过程中可能变得可见。 </li><li>决定样本是否应当被安装在苯甲酸苄酯:苯甲醇（BB:BA，也被称为Murray的清除）或甘油。 注意:我们喜欢更强大的清除剂BB:BA为L. stagnalis胚胎有些不透明。将被安装在BB样品:BA首先需要通过乙醇系列来脱水。将被安装在60％的样品甘油可以立即安装。 </li><li>转移样品到安装溶液:使用移液管（BB BA或60％甘油）中。 注:在安装BB:BA这个一定要在一个玻璃做得好（BB:BA会融化聚碳酸酯塑料）。 </li><li>允许样品以清除5 - 10分钟，然后用叠盖玻片适当数量作为隔离物（1盖玻片样品&lt;1 DPFC，2盖玻片样品&gt; 1 DPFC）它们安装到一幻灯片。 注:所有坐骑现在可以使用与DIC的光学化合物显微镜成像。 </li></ol>

<ol>
	<li>Smith, S. A., Wilson, N. G., Goetz, F. E., Feehery, C., Andrade, S. C. S., 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=Resolving+the+evolutionary+relationships+of+molluscs+with+phylogenomic+tools.">Resolving the evolutionary relationships of molluscs with phylogenomic tools.</a> Nature. 480 (7377), 364-367 (2011).</li><li>Brusca, R. C., Brusca, G. 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> Invertebrates. , (2002).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis:+number+of+people+treated+in+2011.">Schistosomiasis: number of people treated in 2011.</a> Week. Epi. Rec. 88, 81-88 (2013).</li><li>Henry, J. Q., Collin, R., Perry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slipper+snail,+Crepidula.:+an+emerging+lophotrochozoan+model+system.">The slipper snail, Crepidula.: an emerging lophotrochozoan model system.</a> Biol. Bull. 218 (3), 211-229 (2010).</li><li>Perry, K. J., Henry, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+genome+modification+in+the+mollusc,+Crepidula+fornicata.">CRISPR/Cas9-mediated genome modification in the mollusc, Crepidula fornicata.</a> Genesis. 53 (2), 237-244 (2015).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialog+between+genes+and+synapses.">The molecular biology of memory storage: a dialog between genes and synapses.</a> Bio. Rep. 24, 475-522 (2004).</li><li>Jackson, D. J., Ellemor, N., Degnan, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+gene+expression+with+larval+competence,+and+the+effect+of+age+and+parentage+on+metamorphosis+in+the+tropical+abalone+Haliotis+asinina.">Correlating gene expression with larval competence, and the effect of age and parentage on metamorphosis in the tropical abalone Haliotis asinina.</a> Mar. Biol. 147, 681-697 (2005).</li><li>Carter, C. J., Farrar, N., Carlone, R. L., Spencer, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+a+molluscan+RXR+and+evidence+for+its+novel,+nongenomic+role+in+growth+cone+guidance.">Developmental expression of a molluscan RXR and evidence for its novel, nongenomic role in growth cone guidance.</a> Dev. Biol. 343 (1-2), 124-137 (2010).</li><li>Rittschof, D., McClellan-Green, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molluscs+as+multidisciplinary+models+in+environment+toxicology.">Molluscs as multidisciplinary models in environment toxicology.</a> Mar. Pollut. Bull. 50 (4), 369-373 (2005).</li><li>Liu, M. M., Davey, J. W., Jackson, D. J., Blaxter, M. L., Davison, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+set+of+maternal+genes?+Insights+from+a+molluscan+transcriptome.">A conserved set of maternal genes? Insights from a molluscan transcriptome.</a> Int. J. Dev. Biol. 58 (6-8), 501-511 (2014).</li><li>Hohagen, J., Herlitze, I., Jackson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimised+whole+mount+in+situ.+hybridisation+protocol+for+the+mollusc+Lymnaea+stagnalis.">An optimised whole mount in situ. hybridisation protocol for the mollusc Lymnaea stagnalis.</a> BMC Dev. Biol. 15 (1), 19 (2015).</li><li>Raven, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+egg+of+Limnaea+stagnalis.+L.+from+oviposition+till+first+cleavage.">The development of the egg of Limnaea stagnalis. L. from oviposition till first cleavage.</a> Arch. Neth. J. Zool. 1 (4), 91-121 (1946).</li><li>Raven, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+egg+of+Limnaea+Stagnalis.+L.+from+the+first+cleavage+till+the+troghophore+stage,+with+special+reference+to+its'+chemical+embryology.">The development of the egg of Limnaea Stagnalis. L. from the first cleavage till the troghophore stage, with special reference to its' chemical embryology.</a> Arch. Neth. J. Zool. 1 (4), 353-434 (1946).</li><li>Raven, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+in+Limnaea+stagnalis.+and+its+disturbance+by+lithium.">Morphogenesis in Limnaea stagnalis. and its disturbance by lithium.</a> J. Exp. Zool. 121 (1), 1-77 (1952).</li><li>Raven, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+and+origin+of+the+cortical+morphogenetic+field+in+Limnaea.">The nature and origin of the cortical morphogenetic field in Limnaea.</a> Dev. Biol. 7, 130-143 (1963).</li><li>Morrill, J. B., Blair, C. A., Larsen, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulative+development+in+the+pulmonate+gastropod,+Lymnaea+palustris.,+as+determined+by+blastomere+deletion+experiments.">Regulative development in the pulmonate gastropod, Lymnaea palustris., as determined by blastomere deletion experiments.</a> J Exp Zool. 183 (1), (1973).</li><li>Van Den Biggelaar, J. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+the+phases+of+the+cell+cycle+during+the+period+of+asynchronous+division+up+to+the+49-cell+stage+in+Lymnaea.">Timing of the phases of the cell cycle during the period of asynchronous division up to the 49-cell stage in Lymnaea.</a> J. Emb. Exp. Morph. 26 (3), 367-391 (1971).</li><li>Verdonk, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+early+development+of+Lymnaea+stagnalis.">Gene expression in early development of Lymnaea stagnalis.</a> Dev. Biol. 35 (1), 29 (1973).</li><li>Gall, J. G., Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+Detection+of+Rna-Dna+Hybrid+Molecules+in+Cytological+Preparations.">Formation and Detection of Rna-Dna Hybrid Molecules in Cytological Preparations.</a> Proceedings Of The National Academy Of Sciences Of The United States Of America. 63 (2), 378-383 (1969).</li><li>Iijima, M., Takeuchi, T., Sarashina, I., Endo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+engrailed+and+dpp+in+the+gastropod+Lymnaea+stagnalis.">Expression patterns of engrailed and dpp in the gastropod Lymnaea stagnalis.</a> Dev Genes Evol. 218 (5), 237-251 (2008).</li><li>Shimizu, K., Sarashina, I., Kagi, H., Endo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+functions+of+Dpp+in+gastropod+shell+formation+and+shell+coiling.">Possible functions of Dpp in gastropod shell formation and shell coiling.</a> Dev Genes Evol. 221 (2), 59-68 (2011).</li><li>Koop, D., Richards, G. S., Wanninger, A., Gunter, H. M., Degnan, B. M. 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+role+of+MAPK+signaling+in+patterning+and+establishing+axial+symmetry+in+the+gastropod+Haliotis+asinina.">The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina.</a> Dev. Biol. 311 (1), 200-212 (2007).</li><li>Lartillot, N., Lespinet, O., Vervoort, M., Adoutte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+Brachyury+in+the+mollusc+Patella+vulgata+suggests+a+conserved+role+in+the+establishment+of+the+AP+axis+in+Bilateria.">Expression pattern of Brachyury in the mollusc Patella vulgata suggests a conserved role in the establishment of the AP axis in Bilateria.</a> Development. 129 (6), 1411-1421 (2002).</li><li>Jackson, D. J., W&#246;rheide, G., Degnan, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+expression+of+ancient+and+novel+molluscan+shell+genes+during+ecological+transitions.">Dynamic expression of ancient and novel molluscan shell genes during ecological transitions.</a> BMC Evol. Biol. 7 (1), 160 (2007).</li><li>Jackson, D. J., Meyer, N. P., Seaver, E., Pang, K., McDougall, C., 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=Developmental+expression+of+COE.+across+the+Metazoa+supports+a+conserved+role+in+neuronal+cell-type+specification+and+mesodermal+development.">Developmental expression of COE. across the Metazoa supports a conserved role in neuronal cell-type specification and mesodermal development.</a> Dev Genes Evol. 220, 221-234 (2010).</li><li>Perry, K. J., Lyons, D. C., Truchado-Garcia, M., Fischer, A. H. L., Helfrich, L. W., 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=Deployment+of+regulatory+genes+during+gastrulation+and+germ+layer+specification+in+a+model+spiralian+mollusc.">Deployment of regulatory genes during gastrulation and germ layer specification in a model spiralian mollusc.</a> Dev. Dyn. , (2015).</li><li>Iijima, M., Takeuchi, T., Sarashina, I., Endo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+engrailed+and+dpp+in+the+gastropod+Lymnaea+stagnalis.">Expression patterns of engrailed and dpp in the gastropod Lymnaea stagnalis.</a> Dev Genes Evol. 218 (5), 237-251 (2008).</li><li>Shimizu, K., Iijima, M., Setiamarga, D. H. E., Sarashina, I., Kudoh, T., 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=Left-right+asymmetric+expression+of+dpp+in+the+mantle+of+gastropods+correlates+with+asymmetric+shell+coiling.">Left-right asymmetric expression of dpp in the mantle of gastropods correlates with asymmetric shell coiling.</a> EvoDevo. 4 (1), 15 (2013).</li><li>Christodoulou, F., Raible, F., Tomer, R., Simakov, O., Trachana, K., 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=Ancient+animal+microRNAs+and+the+evolution+of+tissue+identity.">Ancient animal microRNAs and the evolution of tissue identity.</a> Nature. 463, (2010).</li><li>Koga, M., Kudoh, T., Hamada, Y., Watanabe, M., Kageura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+triple+staining+method+for+double+in+situ+hybridization+in+combination+with+cell+lineage+tracing+in+whole-mount+Xenopus+embryos.">A new triple staining method for double in situ hybridization in combination with cell lineage tracing in whole-mount Xenopus embryos.</a> Dev Growth Differ. 49 (8), 635-645 (2007).</li><li>Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+fluorescent+in+situ+hybridization+in+the+embryonic+zebrafish+brain+using+differential+detection+systems.">Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.</a> BMC Dev. Biol. 11 (1), 43 (2011).</li><li>Davison, A., Frend, H. T., Moray, C., Wheatley, H., Searle, L. J., Eichhorn, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+behaviour+in+Lymnaea+stagnalis.+pond+snails+is+a+maternally+inherited,+lateralized+trait.">Mating behaviour in Lymnaea stagnalis. pond snails is a maternally inherited, lateralized trait.</a> Biol. Lett. 5 (1), 20-22 (2009).</li><li>Kuroda, R., Endo, B., Abe, M., Shimizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+blastomere+arrangement+dictates+zygotic+left-right+asymmetry+pathway+in+snails.">Chiral blastomere arrangement dictates zygotic left-right asymmetry pathway in snails.</a> Nature. 462 (7274), 790-794 (2009).</li><li>Shibazaki, Y., Shimizu, M., Kuroda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+handedness+is+directed+by+genetically+determined+cytoskeletal+dynamics+in+the+early+embryo.">Body handedness is directed by genetically determined cytoskeletal dynamics in the early embryo.</a> Curr. Biol. 14 (16), 1462-1467 (2004).</li><li>Lu, T. Z., Feng, Z. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sodium+leak+current+regulates+pacemaker+activity+of+adult+central+pattern+generator+neurons+in+Lymnaea+stagnalis.">A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis.</a> PLoS One. 6 (4), e18745 (2011).</li><li>Dawson, T. F., Boone, A. N., Senatore, A., Piticaru, J., Thiyagalingam, S., 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=Gene+Splicing+of+an+Invertebrate+Beta+Subunit+(LCav-beta)+in+the+N-Terminal+and+HOOK+Domains+and+Its+Regulation+of+LCav1+and+LCav2+Calcium+Channels.">Gene Splicing of an Invertebrate Beta Subunit (LCav-beta) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels.</a> PLoS ONE. 9 (4), e92941 (2014).</li><li>Smith, S. A., Wilson, N. G., Goetz, F. E., Feehery, C., Andrade, S. C. S., 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=Resolving+the+evolutionary+relationships+of+molluscs+with+phylogenomic+tools.">Resolving the evolutionary relationships of molluscs with phylogenomic tools.</a> Nature. 480 (7377), 364-367 (2011).</li><li>Gregory, T. R., Nicol, J. A., Tamm, H., Kullman, B., Kullman, K., 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=Eukaryotic+genome+size+databases.">Eukaryotic genome size databases.</a> Nuc. Acids. Res. 35 (Database issue), D332-D338 (2007).</li></ol>

作者什么都没有透露。

这里所描述的方法允许RNA转录物与静水椎实螺的所有发育阶段内想必改变的表达水平的高效率的可视化。为了从它们的胶囊除去胚胎和幼虫我们造模报道其他encapsulated-各种化学，渗透压休克和物理处理的开发模式生物。然而，在我们的手中，我们在这里描述的方法是唯一的高通量技术，消除了艰难的囊膜不破坏胚胎和幼虫。以下解封装，该材料可以被存储，或与蛋白酶K的阶段具体?...

软体动物是一组持有学科的广泛多样化的利益动物。尽管他们的形态多样性1，物种丰富度（仅次于节肢动物的种类数2项）和相关性，以广泛的商业3，4医学和科学问题5-8，也有可以称得上相对较少的种软体动物既装备精良的科学模型，容易在实验室环境维护。即多使用学科，如神经生物学9，生态毒理学10和最近的进化生物学11,12一种软体动物，是静水椎实螺，主要是它的广泛分布和极端易于维护的，因为。尽管它作为一种"模式"有机体的知名度和其悠久的历史使用通过提供给L.发育生物学家13-19，分子工具的范围和功率stagnALIS科学界位于远远落后于更为传统的动物模型（ 果蝇 ，小鼠，海胆，线虫）。 我们的愿望，开发椎实螺的分子模型在指导蛋壳形成的分子机制的兴趣茎。这促使我们缩小一组技术，将允许对椎实螺的发育过程中的基因表达的有效的，一致的和敏感的可视化。 整体原位杂交（WMISH）被广泛用于各种模式生物的和已经使用了超过40年20。在它的不同的形式，ISH可以采用在空间上定位于染色体，rRNA基因，mRNA和微RNA的特异位点。 面临的挑战之一，我们需要解决炼制WMISH方法L.之前stagnalis是温和而有效地提取胚胎和从T不同阶段的幼虫的问题他在它们所沉积的蛋胶囊。该提取或"解封"，需要被有效地实现，以收集足够的物质用于现场试验给出，而在同一时间保持形态和细胞完整性。虽然其他模式生物，也经历了发展封装，在我们手中没有任何报道这些物种的方法可以成功地使用L. stagnalis。 因此该方法的总体目标是:以提取L. stagnalis从它们在高通量胶囊，胚胎和幼虫以施加优化WMISH信号，以提供满意的WMISHsignals用于成像准备胚胎和幼虫预杂交处理。

<table><tbody><tr><td>Featherweight forceps</td><td>Ehlert &amp;amp; Partner</td><td>#4181119</td><td></td></tr><tr><td>Silicon tubing</td><td>Glasger&amp;auml;tebau OCHS GmbH</td><td>760070</td><td></td></tr><tr><td>Glass capillaries</td><td>Hilgenberg</td><td>1403547</td><td></td></tr><tr><td>12 well tissue culture dishes</td><td>Carl Roth</td><td>CE55.1</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Carl Roth</td><td>P733.1</td><td>CAUTION - May cause cancer. Toxic by inhalation, in contact with skin and if swallowed. Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>Carl Roth</td><td>CN06.3</td><td>CAUTION - CAUSES EYE IRRITATION. MAY CAUSE RESPIRATORY TRACT AND SKIN IRRITATION. Avoid breathing dust. Avoid contact with eyes, skin and clothing. Use only with adequate ventilation</td></tr><tr><td>Magnesium Chloride</td><td>Carl Roth</td><td>2189.1</td><td></td></tr><tr><td>Tween-20</td><td>Carl Roth</td><td>9127.1</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Sodium Chloride</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>Ficoll type 400</td><td>Carl Roth</td><td>CN90.1</td><td></td></tr><tr><td>polyvinylpyrrolidone K30 (MW 40)</td><td>Carl Roth</td><td>4607.1</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Nuclease freeBovine Serum Albumin</td><td>Carl Roth</td><td>8895.1</td><td></td></tr><tr><td>Salmon sperm</td><td>Carl Roth</td><td>5434.2</td><td></td></tr><tr><td>Heparin</td><td>Carl Roth</td><td>7692.1</td><td>CAUTION - ADVERSE EFFECTS INCLUDE HEMORRHAGE, LOCAL IRRITATION. POSSIBLE ALLERGIC REACTION IF INHALED, INGESTED/CONTACTED. EYES/SKIN/RESPIRATORY TRACT IRRITANT. POSSIBLE HYPERSENSITIZATION. DURING PREGNANCY HAS BEEN REPORTED TO INCREASE RISK OF STILLBIRTH</td></tr><tr><td>Proteinase-K</td><td>Carl Roth</td><td>7528.1</td><td></td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3790.2</td><td></td></tr><tr><td>Deionised formamide</td><td>Carl Roth</td><td>P040.1</td><td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td></tr><tr><td>Standard formamide</td><td>Carl Roth</td><td>6749.3</td><td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td></tr><tr><td>Triethanolamine</td><td>Carl Roth</td><td>6300.1</td><td>CAUTION - Avoid breathing vapor or mist. Avoid contact with eyes. Avoid prolonged or repeated contact with skin. Wash thoroughly after handling.</td></tr><tr><td>Acetic anhydride</td><td>Carl Roth</td><td>4483.1</td><td>CAUTION - CAUSES SEVERE SKIN AND EYE BURNS. REACTS VIOLENTLY WITH WATER. HARMFUL IF SWALLOWED. VAPOR IRRITATING TO THE EYES AND RESPIRATORY TRACT</td></tr><tr><td>Maleic acid</td><td>Carl Roth</td><td>K304.2</td><td>CAUTION - Very hazardous in case of eye contact (irritant), of ingestion, . Hazardous in case of skin contact (irritant), of inhalation (lung irritant). Slightly hazardous in case of skin contact (permeator). Corrosive to eyes and skin.</td></tr><tr><td>Benzyl benzoate</td><td>Sigma</td><td>B6630-250ML</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. Harmful if swallowed.</td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>10,800-6</td><td>CAUTION - Harmful if swallowed. Harmful if inhaled. Causes serious eye irritation.</td></tr><tr><td>Glycerol</td><td>Carl Roth</td><td>3783.1</td><td></td></tr><tr><td>Blocking powder</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>Anti DIG Fab fragments AP conjugated</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>Tris-HCl</td><td>Carl Roth</td><td>9090.3</td><td></td></tr><tr><td>4-Nitro blue tetrazolium chloride in dimethylformamide&amp;nbsp;</td><td>Carl Roth</td><td>4421.3</td><td>CAUTION - May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes.</td></tr><tr><td>5-bromo-4-chloro-3-indolyl-phosphate</td><td>Carl Roth</td><td>A155.3</td><td>CAUTION - Potentially harmful if ingested. Do not get on skin, in eyes, or on clothing. Potential skin and eye irritant.&amp;nbsp;</td></tr><tr><td>N-acetyl cysteine</td><td>Carl Roth</td><td>4126.1</td><td></td></tr><tr><td>Dithiothreitol</td><td>Carl Roth</td><td>6908.1</td><td>CAUTION - May cause eye and skin irritation. May cause respiratory and digestive tract irritation. The toxicological properties of this material have not been fully investigated.</td></tr><tr><td>Tergitol</td><td>Sigma</td><td>NP40S</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Sodium dodecyl sulphate</td><td>Carl Roth</td><td>CN30.3</td><td>CAUTION - Harmful if swallowed. Toxic in contact with skin. Causes skin irritation. Causes serious eye damage. May cause respiratory irritation.</td></tr><tr><td>Potassium Chloride</td><td>Carl Roth</td><td>6781.1</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Carl Roth</td><td>4984.1</td><td></td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>3904.1</td><td></td></tr><tr><td>Tri sodium citrate dihydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;Na&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Carl Roth</td><td>3580.1</td><td>CAUTION - May cause eye, skin, and respiratory tract irritation. The toxicological properties of this material have not been fully investigated.</td></tr><tr><td>Mineral oil&amp;nbsp;</td><td>Carl Roth</td><td>HP50.2</td><td></td></tr><tr><td>InSituPro-Vsi&amp;nbsp;</td><td>Intavis</td><td></td><td>www.intavis.de/products/automated-ish-and-ihc</td></tr></tbody></table>

a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis

整体原位杂交（WMISH）是一种技术，其允许核酸分子（通常的mRNA）的"整装&#39;组织制剂内的空间分辨率，或发育阶段（例如胚胎或幼虫）的兴趣。 WMISH是非常强大的，因为它可以显著有助于复杂的后生动物基因组的功能特性，正在成为更多的瓶颈与下一代序列数据的泛滥是一个挑战。尽管该技术多的时间，通常需要以优化固有为新颖模型系统WMISH实验的各种参数的概念简单;在组织类型和发育阶段之间的细胞和生化特性的细微差异意味着一个单WMISH方法可能并不适用于所有的情况。我们已经制定了一套产生一致的和重新出现的腹模型静水椎实螺 WMISH方法明确WMISH信号一系列的基因，并在所有发展阶段。这些方法包括未知实足年龄的幼虫到一个个体发育窗口分配，有效地除去胚胎和幼虫从它们的卵胶囊剂，合适的蛋白酶K处理的每个个体发育窗口的应用，和杂交，杂交后和免疫检测脚步。这些方法提供从其中对于给定的RNA转录所得到的信号可以是与探测器专用的调整（主要探针浓度和杂交温度）进一步精制的基础。

在图3所示的代表WMISH染色图案用上面描述的技术产生的，并反映了所涉及的范围内的分子过程，从外壳形成的基因的各种空间表达模式的（ 新基因1，2，3和4），细胞-细胞信号（DPP）在一系列发育阶段的转录调控（ 短尾 ）。虽然我们还没有量化的这些基因，我们期望他们也表达水平变化显著，这表明我们的方法可以对各种各样的处...

Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

整个山<em&gt;原位</em&gt;对腹足软体动物杂交方法<em&gt;静水椎实螺</em

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

a-whole-mount-situ-hybridization-method-for-gastropod-mollusc-lymnaea

Research

JoVE Journal

Biology

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

8.8K 次观看.  Georg-August University of Göttingen. The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

视频: A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

一种非侵入性的方法<em&gt;原位</em&gt;交配成功的女美国龙虾​​（测定<em&gt;美洲螯龙虾</em&gt;）

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

科研

环境科学

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

遗传学

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

免疫与感染

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

发育生物学

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

生物学

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

男性附属性腺产品对腹足软体动物下蛋的影响

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

西普库鲁斯核素体液的原位杂交

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

整个山

摘要

探索更多视频

此视频中的章节

一种非侵入性的方法

全挂载原位杂交法可视化蜱内脏微生物群

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

阿斯蒂亚纳克斯胚胎的全原位杂交

全山原位杂交E8.5，E11.5小鼠胚胎

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

RNA

男性附属性腺产品对腹足软体动物下蛋的影响

西普库鲁斯核素体液的原位杂交

用于老德法纹状体唾液采集的双层膜夹层法

整个山

摘要

探索更多视频

此视频中的章节

一种非侵入性的方法

全挂载原位杂交法可视化蜱内脏微生物群

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

阿斯蒂亚纳克斯胚胎的全原位杂交

全山原位杂交E8.5，E11.5小鼠胚胎

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

RNA

男性附属性腺产品对腹足软体动物下蛋的影响

西普库鲁斯核素体液的原位杂交

用于 老德法纹 状体唾液采集的双层膜夹层法

用于老德法纹状体唾液采集的双层膜夹层法