由国家科学和工程委员会（NSERC），加拿大，加拿大卫生研究院（CIHR）和德Recherche全宗连接桑特杜魁北克省（FRSQ）的资助下进行的工作在Lécuyer实验室的支持。法比奥·亚历克西斯列斐伏尔和盖尔Moquin丽怡NSERC本科生科研奖学金的支持，，而卡罗尔Iampietro所支持的的安杰洛Pizzagalli的博士后奖学金。

1。 RNA探针制备 概述：以下部分介绍了所需要的步骤，使地高辛（DIG）标记的RNA探针适合鱼类。第一步涉及克隆或PCR扩增的序列对应于感兴趣的基因的转录区域，将用于生成一个序列特异性探针。这可以通过以下来实现第一个克隆的基因片段插入的质粒，其中的多个克隆位点的两侧是噬菌体启动子元件（T7，T3或SP6），然后可以使用侧翼结合的启动子序列的引物，作为用于PCR的模板，从而产生与不同的启动子序列，在每个末端（ 图1A）的直链的PCR产物。或者，要避免的克隆步骤，也可以执行PCR扩增，从基因组DNA或cDNA，使用寡核苷酸，包括T7，T3或Sp6启动序列在其5&#39;四肢（ 例如 T7：5&#39;-TAATACGACTCACTATAGGGAGA-3&#39;; T3：5&#39;-AATTAACCCTCACTAAAGGGAGA-3&#39;; Sp6启动：5&#39;-ATTTAGGTGACACTATAGAAGAG-3&#39;）。线性PCR产物为模板，然后使用由运行适当聚合酶体外转录（ 图1B） 中，使高辛标记的正义或反义RNA探针。使特定基因的探针时，我们建议制备义和反义RNA探针的使用，这将是有帮助的，以评估FISH信号的特异性（ 即，除非感兴趣的基因的转录的反义方向中的不同的聚合酶，有义RNA探针将揭示水平的背景信号）。下面的步骤，在所有的人都不应采取非常谨慎，以避免潜在的污染核糖核酸（RNA酶）降解，首先用70％乙醇或商业RNA酶净化解决方案，并清洗所有的板凳区域和设 ​​备，用无RNA酶的用品（ 如：水，提示，微量离心管）。 妈terials <ul><li> 1.5 ml微量离心管（Abgene，罗切斯特，NY，USA猫。0900） </li><li>凝胶提取试剂盒（QIAGEN，密西沙加，ON，加拿大，目录号28706）。 </li><li>无RNA酶的水（欧洲野牛，目录号809-115-CL） </li><li> RNA聚合酶（T7，T3或SP6）。 （20 U /μL，Fermentas公司的生物科技公司。猫。EP0101，EP0111，EP0131）。 </li><li> RNA酶-OUT核糖核酸酶抑制剂（40 U /μl），（Invitrogen公司，伯灵顿ON。Canada.Cat号10777-019）。 </li><li>地高辛（DIG）核苷酸混合物（DIG-11-UTP，罗氏应用生物科学，拉瓦尔，QC，加拿大猫。No.11209256910）。 </li></ul>设备<ul><li>桌面微型离心机</li><li>凝胶电泳装置</li></ul><ol><li> PCR扩增的基因特异性序列的基因组DNA，cDNA或质粒DNA，以产生一个线性的两侧由噬菌体T7，T3或Sp6启动子序列的PCR产物。按照制造商的建议，PCR条件。 </li><li>检查的质量和SI平仄的PCR产物通过琼脂糖凝胶电泳。海关和纯化的PCR产物，使用一个标准的凝胶提取试剂盒，根据制造商建议，以及在50微升缓冲液洗脱产物。 </li><li>通过加入5μl3M乙酸钠（pH 5.2）和150微升冰冷的100％乙醇沉淀出的PCR产物，并将其放置在管，于-70℃下过夜。离心管在13,000 xg离心10分钟，在4℃，并用70％乙醇洗涤沉淀。再次离心，除去乙醇和空气干燥沉淀的痕迹。重悬在25-50微升的无RNA酶的水。 </li><li>转录地高辛标记的探针在20μl反应如下：2μl的10X T7转录缓冲液（Invitrogen公司），1μL（20 U /μl）的RNA酶抑制剂，2μLDIG-NTP混合使用200〜500 ng纯化的PCR产物，和2μl的T7 RNA聚合酶（20单位/微升）。使终体积20微升用无RNA酶的水。转录反应在37℃下温育3-4小时。 </li><li>调节转录ription混合用无RNA酶的水至50μl，并沉淀的地高辛标记的RNA探针在步骤4中所描述的。洗过的RNA沉淀重悬在100μl的无RNA酶的水。再悬浮的样品储存在-70℃下</li><li>量化探头使用的NanoDrop分光光度计。要验证探头质量​​，运行一个1-2微升样品1-2％的琼脂糖凝胶染色，用溴化乙锭。 </li></ol> 2。 果蝇胚胎的收集和固定的 概述：本节介绍的步骤，收获和加工上演了果蝇胚胎。根据所需的胚胎的数目，苍蝇可以维持在人口笼各种尺寸。下面的步骤是对集合进行900厘米3圆柱笼百毫米苹果汁采集板。适当的固定，需要去除保护层，周围的胚胎外绒毛膜内卵黄E透析膜13。一旦收获，在50％的漂白剂溶液以除去绒毛膜胚胎首先沐浴，然后它们在一个含两相溶液的庚烷（permeabilizes卵黄膜），及PBS含有3.7％甲醛混合。卵黄膜，然后裂解除去胚胎转移到一个双相庚烷和甲醇的混合物。适当的胚胎固定和卵黄膜的除去，是为下游FISH程序成功的关键。 物料<ul><li>苹果汁琼脂平板（40％的苹果汁，4％的蔗糖，3.5％琼脂，0.3％甲基对羟基苯甲酸酯）与硬币大小的酵母膏（面包酵母与水混合到黄油的一​​致性） </li><li>粉末状多聚甲醛（电子显微镜科学，哈特菲尔德，PA，USA;货号19208） </li><li>正庚烷</li><li>甲醇</li><li> 3％的漂白粉溶液</li><li> 20毫升的玻璃闪烁瓶（赛默飞世尔科技，渥太华，ON，加拿大猫号03-337-15）。 </li></ul>设备<ul><li>人口笼</li><li>收集筐（使用Nitex网格和切断50ml聚丙烯管中，已被切割的孔在盖子的顶部部分）。 </li><li>小油漆刷</li><li>桌面涡</li></ul><ol><li>预清除以及美联储人口笼子里，与一个新鲜的苹果汁/酵母板1小时，在25°C。 </li><li>添加一个新的苹果汁/酵母片的笼子里收获早期胚胎。孵育笼4小时在25℃（注意，收集时间可以调整，这取决于所需的阶段）。 </li><li>准备通过结合3.7克多聚甲醛粉末，70微升的2N KOH，和7.3毫升的milliQ水中的化学下罩的玻璃闪烁小瓶的新鲜的37％甲醛溶液。煮沸的溶液中，直至粉末完全溶解的多聚甲醛，然后冷却至室温，并过滤器进入新的闪烁瓶中。 </li><li>准备一个双相固定解决方案结合2.25毫升的1X PBS中，用250毫升37％的甲醛，在闪烁瓶中，然后加入7.5毫升的庚烷中。 </li><li>从笼子里收获的苹果汁板，并增加了一点室温的自来水，精心涂刷的油漆刷落盘的胚胎，胚胎收集。再悬浮胚胎转移到一个篮子，不冷不热的自来水冲洗。 </li><li> 90秒Dechorionate胚胎收集洗澡篮子里的漂白粉溶液，然后用清水冲洗彻底，不冷不热的自来水（，直到漂白剂气味没有了）。 </li><li>拆开收集篮，轻轻地用画笔收集胚胎，并将它们传送到双相固定的解决方案。将PBS和庚烷层之间的界面处积累的胚胎。 </li><li>密封闪烁瓶，并固定在旋涡混合器。摇上的最低设置为20分钟。 </li><li>移除并丢弃大部分低级PBS及上层庚烷相，使用巴斯德吸管。吸气剩余的PBS /庚烷/胚胎混合物进入移液管。在滴加的方式，丢弃较低的的PBS阶段从移液器，照顾，以消除胚胎。定金到1.5毫升试管中的双相500μl的庚烷溶液（上层相）和500μl甲醇（下层相）中的胚胎。 </li><li>大力摇晃管手为30-45秒破解的卵黄膜。一旦破裂，胚胎吸收到甲醇。 </li><li>丢弃上正庚烷/甲醇相间庚烷相和未开裂的胚胎，并添加500μL甲醇。摇晃持续5秒。 </li><li>用甲醇洗涤3次，并存储在-20℃（在这一点上的胚胎可以存储为周/月）的胚胎。 </li></ol> 3。固定处理后，杂交，杂交后洗 概述：本节将详细对鱼的胚胎通透的处理步骤之前，预杂交，杂交的RNA探针和杂交后的洗涤液。对于初始的通透性步骤处理的胚胎在单管中作为池（下面的步骤1-9，瞄准为10-15微升结算胚/样品），但它们被分装到一个96孔PCR板的各孔中进行预杂交步骤（下面的步骤10）。这将有助于确保治疗所有的胚胎在初始阶段的实验。设立一个FISH实验时，它是重要的，包括阴性对照以确定个别实验中的背景信号的水平。为此，我们建议，包括“没有探头的样品，并指出在第1节，杂交意识RNA探针，它通常会发出一个信号相媲美的”探针“条件的样本。 材料： <ul><li>甲醇</li><li>磷酸盐缓冲盐水，0.1％Tween-20的（PBT） </li><li>蛋白酶-K 1毫克/毫升原液（Sigma Aldrich公司，奥克维尔，ON，加拿大。产品编号P2308） </li><li>甘氨酸（20毫克/毫升原液） </li><li>杂交溶液：50％甲酰胺，5×SSC，0.1％Tween-20的，100克/毫升的剪切鲑鱼精子DNA，100μg/ ml的肝素。 </li><li> 0.2毫升halfskirted 96孔PCR板，Abgene，罗切斯特，NY，USA猫。没有0900） </li></ul>设备<ul><li>杂交烘箱，数字加热块或水浴可调至56℃，80℃或100℃，或PCR机器。 </li><li>多通道移液器（建议简化洗涤步骤） </li><li>章动搅拌机</li></ul><ol><li>冲洗胚胎在甲醇中，然后在甲醇中的1:1混合物：PBT，然后在PBT的两倍。 </li><li> 20分钟，在1毫升含有3.7％的甲醛（新鲜制备的，如步骤11中所述）的PBT与恒定摆动上nutator修复胚胎。 </li><li>洗3次，每次2分钟，PBT而摇摆的胚胎。 </li><li>准备的溶液为3微克/毫升的蛋白酶K（PK），在PBT稀释，并添加500微升的样品溶液。孵育10分钟，在室温下，无晃动。混合胚胎通过喷射用移液管或轻轻倒转管每2分钟。 </li><li>转移胚胎样品在冰上1小时。 </li><li>移动样品至室温，删除PK溶液。洗2次，持续2分钟，用1ml的PBT + 2毫克/毫升甘氨酸。 </li><li>修复样品20分钟，在1毫升的PBT + 3.7％甲醛与摆动。冲洗胚胎在PBT的5倍。 </li><li>用1毫升的1:1的混合物，PBT和HYB溶液冲洗胚胎。更换用0.5-1毫升100％HYB溶液（在此步骤中的胚胎可以储存在-20℃下的数天/周）。 </li><li>分装胚胎成单个井的96孔板（10-15μl的结算胚胎/孔，照顾到使用较大的光圈提示，以避免破坏胚胎的目的）。 </li><li> HYB溶液100μl/孔样品煮沸5分钟，然后在冰上冷却5分钟。此解决方案是用于样品预杂交（预HYB）。 </li><li>加入100μl/样品预HYB溶液孵育至少2小时，在56°C。 </li><li>对于每个样品，稀释〜100纳克的探针在100微升HYB溶液。热，在80℃下进行3分钟，然后在冰上冷却5分钟（加热探针可以保存在冰中，几个小时）。 </li><li>前HYB的解决方案从胚胎中取出和更换的RNA探针解决方案。 </li><li>杂交，在56℃下进行12-16小时。 </li><li>预温以下的洗涤溶液在56℃下：（A）200微升/样品的HYB溶液;（B）的100微升/样品的3:1混合HYB：PBT，（C）以100μl/样品的1:1混合HYB：PBT，（D）以100μl/ HYB：PBT的1:3混合样品（E）400微升/样本的PBT。 </li><li>冲洗样品用100μl的洗涤溶液A，然后执行洗涤步骤，在56℃用100μl/样品的解决方案的AD，每次15分钟。然后，洗净样品4次5分钟，在56℃下用100μl/样品溶液E </li><li>酷样品至室温。 </li></ol> 概述：这部分描述的抗体和检测试剂，用于检测和可视化的分布的RNA探针信号以下杂交。这些步骤在图2中示意性概述。在这里，我们只给出的标准，我们使用的健壮信号扩增的检测试剂，但存在的各种市售的试剂，可以用来作为替代物，尤其是当执行双-FISH实验合作同时检测不同的RNA，蛋白质-RNA双染色。本协议获得的结果显示在图3 mRNA的表达/本地化图案镶嵌在。 物料<ul><li> HRP共轭的小鼠单克隆抗DIG（Jackson ImmunoResearch Laboratories公司公司猫。编号200-032-156） </li><li>生物素结合的小鼠单克隆抗-DIG（杰克逊IMMUNoResearch安全检测实验室公司猫。号200-062-156） </li><li>链霉亲和素-HRP结合物（分子探针，尤金OR，USA，大类No.S991） </li><li> CY3-酪胺结合物（Perkin Elmer公司生命科学学院，波士顿，MA，USA，猫号SAT704A） </li><li> DABCO安装解决方案：在一个50毫升的试管中，稀释的1.25克DABCO（1,4 - 二氮杂双环[2.2.2]辛烷;度Sigma D-2522），在15毫升的1X PBS中，然后加入35毫升甘油并混合直到完全均匀。通过预混合，用PBS，DABCO粉末溶解非常迅速。在-20°C的商店安装解决方案</li><li>载玻片，盖玻片</li></ul>设备<ul><li>章动搅拌机</li><li>多通道移液器</li><li>荧光显微镜</li></ul><ol><li>准备PBTB溶液，含有PBT + 1％无脂干牛奶（通常为50-100毫升是足以满足所有的检测试剂孵育和洗涤步骤）。 </li><li>块样品在100的微升PBTB /样品在室温下10分钟而摇摆nutator。 </li> &lt;李&gt;删除阻塞的解决方案，从胚胎和孵化样品在室温下用100微升/样品的单克隆生物素的抗DIG抗体溶液（稀释1/400现货成PBTB），与摇摆1.5-2小时。 </li><li>洗净样品6次，每次5分钟用100μlPBTB的/样品，与摆动。 </li><li>在室温下孵育1.5小时，用75毫升/样品的链霉亲和素-HRP溶液（1/100稀释库存在PBTB，10μg/ ml的最终浓度）与摆动。 </li><li>洗净样品5分钟6次各100μlPBTB的/样品与摆动（为的DNA counterstaining，稀释的DAPI 1X工作在PBTB浓度和使用这种解决方案，在第一清洗工序中）。 </li><li>的胚胎冲洗与PBT，然后5分钟，PBS洗2次。 </li><li>孵育2小时，在室温下对样品用50μl/样品的Cy3酪胺的溶液（稀释1/50酪胺扩增缓冲液中）的nutator。从这个步骤上，一定要保持样品的光屏蔽插座。/ P&gt; <li>洗净样品6次，用100μl的PBS /样品上nutator每次5 min。 </li><li>移除PBS及添加100-125微升/安装解决方案的样品含有70％甘油和2.5％（DABCO）。将样品在4°C。这些样本可以被存储了好几年。 </li><li>使用一个大孔的前端，轻轻移液管的样品悬浮胚胎转移胚胎到玻璃载片上的25微升，然后放置在胚胎和密封到与指甲油滑动盖玻片。 </li><li>分析胚胎样品用荧光显微镜。 </li></ol>

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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=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445, 168-176 (2007).</li><li>Lecuyer, E., 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=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Imai, K. S., Levine, M., Satoh, N., Satou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+blueprint+for+a+chordate+embryo.">Regulatory blueprint for a chordate embryo.</a> Science. 312, 1183-1187 (2006).</li><li>Bell, G. W., Yatskievych, T. A., Antin, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEISHA,+a+whole-mount+in+situ+hybridization+gene+expression+screen+in+chicken+embryos.">GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos.</a> Dev. Dyn. 229, 677-687 (2004).</li><li>Wilkie, G. S., Shermoen, A. W., O'Farrell, P. H., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcribed+genes+are+localized+according+to+chromosomal+position+within+polarized+Drosophila+embryonic+nuclei.">Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei.</a> Curr. Biol. 9, 1263-1266 (1999).</li><li>Rothwell, W. F., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+embryo+dechorionation.">Drosophila embryo dechorionation.</a> CSH Protoc. , (2007).</li><li>Stapleton, M., 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=The+Drosophila+gene+collection:+identification+of+putative+full-length+cDNAs+for+70%+of+D.+melanogaster+genes.">The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes.</a> Genome Res. 12, 1294-1300 (2002).</li><li>Rubin, G. M., 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=A+Drosophila+complementary+DNA+resource.">A Drosophila complementary DNA resource.</a> Science. 287, 2222-2224 (2000).</li><li>Tomancak, P., 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=Systematic+determination+of+patterns+of+gene+expression+during+Drosophila+embryogenesis.">Systematic determination of patterns of gene expression during Drosophila embryogenesis.</a> Genome Biol. 3, (2002).</li><li>Terashima, J., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+available+food+into+the+number+of+eggs+laid+by+Drosophila+melanogaster.">Translating available food into the number of eggs laid by Drosophila melanogaster.</a> Genetics. 167, 1711-1719 (2004).</li><li>Lecuyer, E., Parthasarathy, N., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+protocols+in+Drosophila+embryos+and+tissues.">Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.</a> Methods Mol. Biol. 420, 289-302 (2008).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fluorescent+in+situ+hybridization+in+Drosophila.">High resolution fluorescent in situ hybridization in Drosophila.</a> Methods Mol. Biol. 714, 31-47 (2011).</li><li>Kosman, D., 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=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846 (2004).</li></ol>

没有利益冲突的声明。

探针的合成步骤，我们通常会产生径流的反义RNA探针， 通过体外转录全长果蝇的cDNA扩增出质粒发现在果蝇的基因收藏（DGC），资源详载于以下网址： <a href="http://www.fruitfly.org/DGC/index.html" target="_blank">http://www .fruitfly.org / DGC / index.html的</a> 14,15的 。这种方法已被广泛使用在的大型原位杂交研究，目的是在果蝇胚胎9,16测绘基因的表达和mRNA的定位模式。 DGC的质粒?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> T7，T3或SP6 RNA聚合酶</td><td> Fermentas公司的生命科学</td><td> EP0101，EP0111，EP0131 </td><td>套件包含反应缓冲液。 </td></tr><tr><td> DIG RNA标记液</td><td>罗氏应用科学部</td><td> 11 277 073 910 </td><td></td></tr><tr><td> RNAguard </td><td> Amersham Biosciences公司</td><td> 27-0816-01 </td><td></td></tr><tr><td> 3M醋酸钠</td><td></td><td></td><td></td></tr><tr><td>冷的100％乙醇。 </td><td></td><td></td><td></td></tr><tr><td>冷的70％乙醇。 </td><td></td><td></td><td></td></tr><tr><td>含氯漂白剂稀释液与水1:1。 </td><td></td><td></td><td></td></tr><tr><td>正庚烷</td><td></td><td></td><td></td></tr><tr><td>甲醇</td><td></td><td></td><td></td></tr><tr><td>蛋白酶K </td><td> Sigma Aldrich公司，ON，加拿大奥克维尔</td><td>产品编号P2308 </td><td></td></tr><tr><td> 40％的甲醛溶液，新鲜制备的</td><td></td><td></td><td></td></tr><tr><td> PBS-Tween溶液（PBT）的</td><td></td><td> 1xPBS，0.1％Tween-20的</td><td></td></tr><tr><td>甘氨酸溶液</td><td></td><td> 2毫克/毫升在PBT的甘氨酸</td><td></td></tr><tr><td> HRP共轭的小鼠单克隆抗DIG </td><td>杰克逊ImmunoResearch Laboratories公司</td><td> 200-032 - 156 </td><td> （1/400稀释的1毫克/毫升原液PBTB </td></tr><tr><td> HRP偶联的羊的单克隆抗DIG </td><td>罗氏应用王欧，吴文良CE，拉瓦尔，QC </td><td> 1 207 733 </td><td> 1/500稀释的原液PBTB </td></tr><tr><td>生物素结合的小鼠单克隆抗-DIG </td><td>杰克逊ImmunoResearch Laboratories公司公司，西部树丛，PA，USA </td><td> 200-062-156 </td><td> （1/400稀释的1毫克/毫升原液PBTB </td></tr><tr><td>链霉亲和素-HRP结合物</td><td>分子探针，尤金OR，USA </td><td> S991 </td><td> （1微克/毫升的库存中的1/100稀释的</td></tr></tbody></table>

whole mount rna fluorescent in situ hybridization of drosophila embryos

评估的表达模式的基因，以及其转录RNA的亚细胞定位性能，了解其生物学功能在开发过程中的关键功能。 RNA 原位杂交（RNA-ISH）是一个功能强大的方法，用于可视化的RNA分布特性，无论是在有机体，细胞或亚细胞水平的1。 RNA-ISH基于杂交的标记的核酸探针（ 例如，反义RNA，寡核苷酸）互补的mRNA的序列，或一个非编码RNA靶景点2。由于该过程要求单独初级序列信息来生成序列特异性探针，它可以普遍适用于范围广泛的生物体和组织标本3。事实上，一些大型ISH研究已实施的记录在各种模式生物的基因表达和RNA的本地化动态，这导致了重要的社会资源，建立4-11。虽然多年来，已开发各种探针标记和检测策略的结合使用荧光标记的检测试剂和酶的信号放大步骤提供显着增强的灵敏度和分辨率的程序12。在这里，我们描述了一个优化的荧光原位杂交法（FISH）采用酪胺信号放大（TSA）中上演了果蝇胚胎的RNA表达和定位动态可视化。该过程进行96孔PCR板格式，极大地方便了同时处理大量的样品。

当成功执行，这个程序提供了一个在果蝇胚胎发育早期的基因表达和mRNA的定位动态的时空分析的详细程度显着增强。事实上， 如图3A所示为经典的配对规则基因欠幅脉冲 （ 运行 ），可以使用这个协议来观察基因表达事件通过检测组新生成绩单灶的表达核。此外，如胚胎镶嵌在图3B中所示，该方法能够在高分辨率的mRNA的定位功能的可视化。 <p class=...

Watch this Scientific Journal Video about Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos at JoVE.com

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

在这里，我们描述了一个全型日光灯<em在原位</em杂交（FISH）协议确定的RNA表达在果蝇胚胎发育过程中的表达和定位性​​能，<em&gt;果蝇</em&gt;。

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

whole-mount-rna-fluorescent-situ-hybridization-drosophila

Research

JoVE Journal

Biology

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

18.2K 次观看.  Institut de Recherches Cliniques de Montréal (IRCM). 在这里，我们描述了一个全型日光灯

视频: Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

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

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

使用全山原位杂交分子和有机体生物学链接

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

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 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...