Name: fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues
Uploaded: 2014-02-24T15:30:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

研究在加尼姆实验室是由研究经费不支持。 908-42.12/2006从德国和以色列基金会（GIF），不授予。 IS-4062-07从美国和以色列两个民族组成的农业研究和发展基金（BARD），以及研究经费没有。 884/07由以色列科学基金会（ISF），以MG

1。一般白粉虱，植物和病毒的准 ​​备FISH分析<ol><li>对棉花幼苗（ 陆地棉品种。阿卡拉）后B和Q型烟粉虱和里面的25±2℃，相对湿度60％标准条件下防虫笼和增长间保持，和一个14小时光照/ 10小时黑暗的光照。 </li><li>进行PCR与利用共生细菌特异性引物如前所述15-19内共生菌感染测试。 </li><li>从商业幼儿园或植物番茄种子购买番茄苗（ 期Zn2 +茄品种。牛排）。 </li><li>与上述相同的饲养条件下保持，直到植物达到4真叶阶段，患者的年龄为粉虱介导的接种TYLCV 15的最合适的（见第4.1段）。 </li></ol> 2。驱虫处理，固定，探针设计，杂交和可视化远藤鱼共生体<ol><li>收集10个成人粉虱从棉花植株吸出，或选择10 第 3或第 4龄若虫从棉花的叶子。 </li><li>立即浸入冷水中卡诺固定液（氯仿 - 乙醇 - 冰醋酸，6点03分01秒，体积/体积）的Eppendorf管内。定为2小时至过夜（最好过夜）。 </li><li>彻底去除固定剂和脱色的6％H 2 O 2在乙醇2小时。 </li><li>保留几天的无水乙醇的样品至数周，在室温下或继续到下一个步骤。 </li><li>设计的探头根据每个细菌的16S核糖体RNA基因的反向互补的DNA引物。对于设计的探头，考虑到该帐户的同样的考虑来设计PCR引物。使用寡核苷酸探针BTP1 5&#39;-Cy5的-TGTCAGTGTCAGCCCAGAAG-3&#39;，Rb1的5&#39;-Cy3标记TCCACGTCGCCGTCTTGC-3&#39;，BTH 5&#39;-Cy3标记CCAGATTCCCAGACTTTACTCA-3&#39;和ARS2 5&#39;-Cy3标记TCATGACCACAACCTCCAAA-3&#39;为Portiera，立克次体，Hamilonella和Arsenophonus，分别具体目标。 </li><li>每毫升过夜杂交的样品在黑暗中在杂交缓冲液（20mM的Tris-HCl，pH值8.0，0.9 M氯化钠，0.01％[重量/体积]十二烷基硫酸钠，30％[体积/体积]甲酰胺）中含有10皮摩尔荧光探针。如果有必要结合一个以上的探针用不同的荧光染料。使用未感染粉虱与靶细菌和无探针的样品作为阴性对照。 </li><li>装载样品整体，在显微镜载玻片上，在包含有液体阻滞剂杂交缓冲液，盖上盖玻片，用密封指甲油，并根据荧光或共聚焦显微镜视图。 </li></ol> 3。粉虱器官解剖，固定，杂交和可视化TYLCV鱼<ol><li>收集成人粉虱由愿望和ANE持续饲养TYLCV感染的番茄植株通过暴露于纸巾浸渍丙酮sthetize。揭露粉虱只有1-2分钟太长时间暴露在丙酮中可以修复粉虱组织。用饲养在被感染的番茄植株，无探头样品对照nonviruliferous粉虱，以确认具体的杂交。 </li><li>对抑郁症的显微镜山粉虱幻灯片，使用立体显微镜器官解剖。 </li><li>从前胸拉开昆虫头部和解剖唾液腺在1x PBS中添加1％甲苯胺蓝染色进行更好的可视化，由于其体积小。允许的色斑吸收2-3分钟。 </li><li>在胸部和腹部，观察并驱逐出肠之间的交界处拉虫分开。通过与上腹部尖镊子推驱逐腹部的内容。 </li><li>轻轻地从成功的解剖取出PBS和甲苯胺蓝，轻轻地加入300微升卡诺固定液固定器官5分钟。 </li><li>一从凹陷以及一个侧面而不是从顶部西元固定剂，以防止解剖器官的失控的移动。一旦触摸固定液，该机关将坚持以玻璃，并在整个过程中，他们不会移动。 </li><li>除去固定液，加入500微升杂交缓冲液补充有10皮摩尔的荧光DNA探针的Cy5-5&#39;-GGAACATCAGGGCTTCGATA-3&#39;，它互补于在TYLCV外壳蛋白基因的序列组成。 </li><li>通过孵育滑动的小湿润室用湿毛巾纸在下面组成的小塑料盒内的样品杂交过夜，在室温下在黑暗中。 </li><li>继杂交，拿起机关用细解剖工具，并迅速将其移至含有30微升的新的杂交缓冲液补充用DAPI（0.1 mg / ml的1×PBS中），并包含有液体阻滞剂新鲜显微镜幻灯片。 </li><li>盖上盖玻片的标本，密封机智ħ指甲油和视域下的荧光或共聚焦显微镜。 </li></ol> 4。厂处理，手工切片，固定和杂交的鱼TYLCV <ol><li>使用带毒粉虱介导的接种接种番茄幼苗TYLCV。可以用接种后一周无症状植物这种鱼方法检测病毒。典型的疾病的症状是明显的后三周接种。使用被感染的番茄植株也没有探测样品对照，以确认具体的杂交。 </li><li>使用锋利的组织学刀片编制番茄手切2-4厘米长的纵向或横截面的茎和叶。 </li><li>在Eppendorf管中，固定在卡诺固定液的章节2小时至室温过夜。 </li><li>除去固定液和保存在无水乙醇中的章节数天至数周，在室温下或继续到下一个步骤。 </li><li>洗净切片三次，每次1分钟，在杂交缓冲液。 </li><li>杂交的部分用500μl的杂交缓冲液中添加有10皮摩尔的荧光寡核苷酸探针的Cy3-5&#39;-GGAACATCAGGGCTTCGATA-3&#39;，它互补于TYLCV外壳蛋白基因的序列组成。 </li><li>冲洗切片3次，每次1分钟，在杂交缓冲液中。 </li><li>山路段全在杂交缓冲液补充用DAPI（0.1 mg / ml的1×PBS中），并包含有液体阻断剂，盖上盖玻片，用密封在荧光或共聚焦显微镜指甲油和看法。 </li></ol>

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此处描述的植物病毒在其宿主植物和昆虫载体的本地化和共生细菌在其特定粉虱主机协议，可适于其它病毒在植物中，甚至在动物组织中的定位。此外，该协议可以用于本地化共生和致病性细菌和在植物和动物的系统的其他微生物。所描述的方法依赖于杂交的一个短的，荧光标记的寡核苷酸DNA探针与靶DNA或RNA分子在细胞之间简单的概念。的结果是一个特定的杂交，以最小的背景，并使用荧光或共聚焦显微镜检测的信号。几个探针用不同的荧光染料，针对一个以上的基因，可以同时使用。这使得可视化多个目标中的单个细胞或组织。在这些协议中所述的程序很简单，样本处理时间是最小的。与其它的协议，如高背景信号，长时间处理试样和使用大量和昂贵的材料，用于分析有关的问题，并不限制在这里所描述的协议。 B的B型和Q生物型粉虱粉虱被用来描述协议在这个手稿，但是，饲养条件适用于任何粉虱生物型，各自的寄主植物。粉虱完成其生命周期中所指出的条件下三周。使用的B型人口的每一个人被感染的主要共生细菌Portiera，二次内共生菌Hamiltonella和立克次体的 Q型人被感染Portiera和Arsenophonus。世界各地的其他粉虱种群被证明感染了这些或其它共生细菌种类，并具有不同空间localizati关于在体内16-20图案。 TYLCV感染的植物显示出典型的疾病症状后接种三周。对TYLCV定位，对症植物可用于病毒采集由粉虱24小时和病毒可在感染的植物和昆虫使用上述FISH协议被检测到。而在昆虫中使用的探针可以结合有许多荧光团可与不同波长的光，包括Cy3和Cy5切除，在植物中的Cy3是最适合的，并且不发出荧光，在类似的波长为叶绿体，在植物细胞中最丰富的细胞器。 成功执行的细菌和植物和昆虫病毒的定位所描述的协议依赖于样品的良好固定为更好的可视化和探针的渗透，良好的探头设计为特异性和用H在整个昆虫的情况下进行结算2 O 2 b埃特信号的可视化。在FISH在植物的情况下，手工制作部分必须尽可能薄为好探头穿透力和更好的可视化的信号。这可以通过把叶2泡沫塑料片之间，以帮助切割，并通过使用专业的刀片，可以从多个供应商处购买用于此目的加以改进。 该协议是不适合的目标的亚细胞定位，但它有可开发的潜力，使用探头格式在这里描述的，在一个合适的方法的亚细胞定位。例如，此处所描述的短探针可以缀合到分子如生物素，而不是荧光分子，这反过来又可以使用缀合到链霉亲和金颗粒，可以在TEM下进行可视化作为目标。这以后的修改可以适用于多个目标，包括基因转录和微生物的亚细胞定位。最后，所描述的协议s为适于与核酸如DNA和RNA，但不杂交蛋白。 用于获得在此描述的FISH协议的最佳结果，最好是使用新鲜的昆虫和植物材料，以及新鲜固定液和杂交缓冲液。该探测器应始终保持在-20℃的小等份，以避免反复冻融。每个所述步骤的处理时间可以根据所研究的生物体进行校准。诚如协议，以下固定和脱色，粉虱和植物样品可在室温下保存很长一段时间的无水乙醇。发送时的样品进行处理从一个位置到另一个位置，或执行长时程实验时，这一步骤是特别有用的。杂交信号的质量不受影响以下这样长时间保存。

当研究其感染的植物宿主植物病毒和其他病原体之间的相互作用，它以可视化的病原体和它们各自的核酸在原位 ，而不论它们是否会造成对寄主的负面影响是非常重要的。内部和之间的植物细胞研究病原体的运动时，这是最重要的。 在病原体的基因产物原位定位是补充其他方法为研究致病过程中的重要一步。许多植物病原菌，尤其是病毒，是由昆虫传播的，有其复杂的向量和亲密的互动。这些病毒在它们的矢量定位，是研究的路径进行传输重要的，并且该载体内的可能的相互作用位点。某些昆虫向量植物病毒的传播是通过驻留这些昆虫内共生菌资助。为了更好地研究这些植物病毒B中的传输Ÿ他们的向量，它也是必不可少的，以可视化的一般内共生菌，以及参与病毒传播，尤其如此。因此，病毒和内共生细菌的共存需要调查在其昆虫宿主这些生物之间的可能关系。除了病毒传播，内共生细菌的影响昆虫媒介生物学等几个方面，这些细菌内昆虫因而空间定位较高的兴趣和重视的。 番茄黄化曲叶病毒 （TYLCV）（菜豆金色花叶病毒，Geminiviridae）是栽培番茄的1-4全世界最重要的病毒性疾病复杂。 TYLCV是一种韧皮部限制病毒和专门向量由粉虱 5,6。模型描述其向量粉虱双生的易位已经提出了6-9。两个具体的障碍正在积极越过DURing这个循环式传输:中肠/血淋巴和血淋巴/唾液腺障碍。这个传输过程中被假设由未知的受体能识别病毒衣壳来介导的。 TYLCV被认为是穿越B。粉虱肠6,10，并通过主唾液腺6吸收之前它被注入到植物。在粉虱血淋巴，TYLCV与由昆虫次级内共生菌Hamiltonella产生的GroEL的蛋白相互作用。这种相互作用保证了在血淋巴TYLCV的安全传输，并且由昆虫免疫系统11。Hamiltonella和Portiera，初级共生细菌免受攻击保护它粉虱的，都装在bacteriocytes，血淋巴和房子内共生菌11。B.发现昆虫细胞粉虱怀有额外的内共生菌，包括立克次氏体，Arsenophonus，沃尔巴克氏体和弗里奇EA，它可以内部或外部的bacteriocytes是局部的，并且对昆虫的生物学12不同的影响。 若干报告已经尝试通过使用费时和昂贵的协议，如透射电子显微镜（TEM），抗体和RNA 原位上微观厚部10,13来研究TYLCV的植物和粉虱定位，一项最近的研究中所述的定位通过使用一个简单的协议，14他们的工厂和矢量主机内的植物病毒。在这里，我们描述了一个简单的协议TYLCV在B中的定位烟粉虱解剖肠和唾液腺，并在部分从TYLCV感染的植物编制。我们进一步描述Portiera，B的主要共生细菌的本地化烟粉虱，其二次内共生菌Hamitonella，立克次氏体和Arsenophonus，该协议是基于使用短的DNA探针，即荧光标记在其5&#39;末端，并特异性杂交​​中病毒或细菌的基因序列互补的序列。样品处理是比较容易和得到的信号是高度特异性的。所描述的协议可以用来在他们的植物，动物和昆虫宿主本地化的病毒，细菌和其它病原体，并能进一步用于在任何给定的组织定位的mRNA。

<table><tbody><tr><td>Fluorescently labeled&amp;nbsp; Probes</td><td>Metabion</td><td>20 bp HPLC purified</td><td>Sequence designed by customer</td></tr><tr><td>Toluidine blue</td><td>Sigma-Aldrich</td><td>89640</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>Molecular Biology Grade</td></tr><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>F9037</td><td>Molecular Biology Grade</td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td>Molecular Biology Grade</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Molecular Biology Grade</td></tr><tr><td>Liquid Blocker</td><td>Ted Pella Inc.</td><td>22309</td><td></td></tr></tbody></table>

fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues

荧光原位杂交（FISH）是考虑到了各种常用的用于可视化基因转录物在真核细胞中，并且可以被进一步修饰以可视化单元中的其他组分，如感染病毒和细菌的方法的名称。空间定位和感染过程中病毒和细菌的可视化的是，在应对不同的刺激补充表达谱实验，如芯片和RNAseq一个必不可少的步骤。了解时空感染这些药物补充生物实验旨在了解与细胞成分及其相互关系。几种技术用于可视化病毒和细菌等的报告基因系统或免疫组织化学的方法是耗时的，并且一些被限制与模型生物体的工作，并涉及复杂的方法。 FISH靶向的RNA或DNA的物种的细胞是相对容易和快速的我的ThOD用于研究基因的时空定位和诊断的目的。此法可强健和相对容易实现，当协议使用短的杂交，商业购买的探头，它并不昂贵。这是特别可靠时的样品制备，固定，杂交和显微可视化不涉及复杂的步骤。在这里，我们描述了一个协议，用于细菌和病毒在昆虫和植物组织定位。该方法是基于简单的制剂，固定，和昆虫整个支架并解剖器官或手工制作的植物部分杂交，用20个碱基对缀合的荧光染料在其5&#39;或3&#39;短的DNA探针末端。这个协议已经被成功地应用于许多昆虫和植物组织，可用于分析在细胞中的mRNA或其它RNA或DNA的物种中的表达。

研究了在该手稿的系统示于图1，并包括一个受感染的植株用TYLCV，成年人和粉虱B的若虫粉虱和白粉虱示出了用于在昆虫TYLCV易位路径的内部解剖结构， 图2示出了双FISH在成年烟粉虱的初级共生Portiera和次级共生Arsenophonus。 图3显示了双FISH为Portiera和Hamiltonella，并图4显示了双鱼的Portiera和立克次体，这两个数字在B。粉虱 第 4龄若虫， 图 5示出了用于FISH TYLCV和DAPI染色的粉虱解剖肠，和图6显示FISH对TYLCV和DAPI染色的解剖粉虱初级唾液腺， 图7示出了用于FISH TYLCV和DAPI染色在手工切割TYLCV感染的植物部分。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig1highres.jpg" src="/files/ftp_upload/51030/51030fig1.jpg" /> 图1。研究了这篇稿子系统的总体概述A）引起的感染番茄黄化曲叶病毒（TYLCV），B）典型疾病症状的成年粉虱的4 个若虫期的。 三）外部视图的外形图在烟粉虱发育的生命周期。其他若虫阶段是在一般的外部视图相似，但尺寸较小。D）一种成人B的内部解剖结构的示意图粉虱表示TYLCV采集从植物筛元件，流通和传输（黑色箭头）的路径。红色颗粒参阅TYLCV病毒粒子。号码:韧皮部，S:S<html> tylet，E:食管; FC:过滤室; DM:降肠;是:升中肠，CA:盲肠，HG:肠; BC:bacteriocytes; PSG:主要唾液腺<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51030/51030fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig2highres.jpg" src="/files/ftp_upload/51030/51030fig2.jpg" /> 图2。 Portiera和Arsenphonus对B的双鱼烟粉虱 Q型成人（A）和放大上标示的区域（B）两者在明亮的领域和渠道相结合的光学部分通过检测每个探针。Portiera 特异性探针（红色）结合到Cy5的和Arsenphonus特异性探针（ 发射的荧光信号黄色）缀合Cy3的被使用。 E:鸡蛋。PG"目标="_blank"&gt;点击这里查看大图。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig3highres.jpg" src="/files/ftp_upload/51030/51030fig3.jpg" /> 图3。Portiera和Hamiltonella对B.双鱼烟粉虱若虫。Portiera 特异性探针（红色）结合到Cy5的和Hamiltonella特异性探针（绿色）结合到Cy3的被使用。从组合的光学部分获得下暗场观察。B A）Hamiltonella杂交信号）从获得Portiera杂交信号结合光学部分和下暗场观察。C）Portiera和Hamiltonella合并信号在明亮的领域。下哒观察D）Portiera和Hamiltonella组合信号RK场。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51030/51030fig3highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig4highres.jpg" src="/files/ftp_upload/51030/51030fig4.jpg" /> 图4。Portiera和立克次氏体对B.双鱼烟粉虱若虫。Portiera 特异性探针（红色）结合到Cy5的和立克次氏体特异性探针（蓝色）结合到Cy3的被使用。 一 ）从组合的光学部分获得下暗场观察立克次体的杂交信号:B从获得）Portiera杂交信号在明场相结合的光学部分和下暗场观察。C）Portiera和立克次体组合信号D） <eM&gt; Portiera和立克次体在暗场相结合的信号。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51030/51030fig4highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig5highres.jpg" src="/files/ftp_upload/51030/51030fig5.jpg" /> 图5。鱼对TYLCV成人B的解剖肠这已经从被感染的番茄。TYLCV特异性探针（红色）结合到Cy3的收购病毒48小时粉虱女性使用，并且安装和可视化。 一 ）解剖肠从合并为见过的中肠是用DAPI染色在亮视野光学部分B）TYLCV FISH信号（红色）和DAPI染色的细胞核（蓝色）从下暗场组合的光的部分看到。 FC:滤室; DM:descendi<html>吴肠;是:升序肠，CA:盲肠，HG:肠。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51030/51030fig5highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig6highres.jpg" src="/files/ftp_upload/51030/51030fig6.jpg" /> 图6。在成人B的解剖主要唾液腺鱼的TYLCV这已经从被感染的番茄。TYLCV特异性探针（红色）结合到Cy3的收购病毒48小时粉虱女性使用，而唾液腺安装和可视化。 一 ）解剖和DAPI染色主要唾液腺前用DAPI染色从下明场B）TYLCV FISH信号（红色）和DAPI染色的细胞核（蓝色）从下暗场结合部分看去结合部分腺看去。s :/ / www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51030/51030fig6highres.jpg"目标="_blank"&gt;点击这里查看大图。 <img alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig7highres.jpg" src="/files/ftp_upload/51030/51030fig7.jpg" /> 图7。通过TYLCV感染的植物的茎鱼TYLCV手切纵截面。TYLCV特异性探针（红色）结合到Cy3的使用，并结合DAPI染色安装和可视化。A）和红色显示的一个部分之前TYLCV信号在韧皮部筛分子，和DAPI染色的细胞核从下暗场B）结合部分在亮视野的方向在（A）中所示的同一个组合的部分看到。 pH值:韧皮部; XY:木质部<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51030/51030fig7highres.jpg" target="_blank">点击这里查看大IM</a>年龄。

Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

荧光原位杂交 FISH 用于植物和昆虫组织中病毒和内共生细菌的定位免疫学和感染|视频

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

我们在这里描述原位杂交简单的荧光（FISH）方法对病毒和细菌的昆虫和植物组织的定位。这个协议可以延长mRNA的整装可视化和显微切片。

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

fluorescence-situ-hybridizations-fish-for-localization-viruses

Universidad San Sebastian

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JoVE Journal

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Biology

Immunology and Infection

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Genetics

Viruses

SCIENTISTS IN ACTION

23.4K 次观看.  Volcani Center. 我们在这里描述原位杂交简单的荧光（FISH）方法对病毒和细菌的昆虫和植物组织的定位。这个协议可以延长mRNA的整装可视化和显微切片。 

视频: Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

科研

免疫与感染

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

生物学

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

神经科学

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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 ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

荧光

摘要

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荧

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色

蛋白质中 Plasmodesmal 定位序列的识别在底中

原位杂交技术在植物中的精确定位成绩单

荧光检测病毒RNA

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

荧光

摘要

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此视频中的章节

荧

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

蛋白质中 Plasmodesmal 定位序列的识别在底中

原位杂交技术在植物中的精确定位成绩单

荧光检测病毒RNA

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染