Name: hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes
Uploaded: 2012-06-28T12:00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes at JoVE.com

作者想感谢在发展杂交意见玛丽卡·沃尔特斯的协议进行讨论冈比亚按蚊的胚胎，随后调整和修改开发协议， 在这里介绍杂交原位杂交在软组织和尤里Goltsev的原位方法伊蚊和致倦库蚊胚胎原位 。我们也承认亚当·PARE和大卫Kosman的有益建议。我们感谢奥斯瓦尔多Marinotti科学讨论和编辑的协议文本。

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作者有没有披露。

提出了整个安装蚊子组织和胚胎在原位和色度染色协议的杂交，是一个有用的技术，在特定器官和细胞类型的转录本地化。这些程序是一个比我们以前报道的方法，无论是在精简广泛的洗涤步骤，并提供更多的技术细节和试剂来源的改善。 根据我们的经验，比色法检测杂交信号杂交信号的灵敏度和清晰度优于基于荧光检测计划。此外比色法检测绕开了胚胎，这是本质上的自动荧光信号歧视的问题。检测杂交信号的局限性发生时，低丰度转录的针对性和背景染色是显而易见的。杂交温度提高到65°C已被发现，以减少回地面的杂交信号，但并不是唯一的目标特定的RNA探针设计的建议替代。 该协议已被用来执行整个安装唾液腺，埃及伊蚊和冈比亚按蚊，按蚊，AE的卵巢和胚胎原位杂交。埃及斑蚊和致倦库蚊 。这种方法也适用于其他蚊虫的组织，想必其他昆虫。此外，我们还首次为三个病媒蚊，AE在胚胎发育分期的比较准则编制。埃及斑蚊，安。冈比亚和CX。 fatigans。谨慎饲养条件下，这三个品种的特定菌株已报告的意见。重要的是要注意，不同蚊种的不同菌株，不同饲养条件下发育的时间当然可以。 在原位补充杂交持续努力向全日空lyze蚊子和其他节肢动物的转录，并可能在这些微生物的基因表达调控提供更好的画面。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 0.5 M的EDTA </td><td> Ambion公司</td><td> AM9261 </td><td></td></tr><tr><td> 1M的Tris-盐酸</td><td> Ambion公司</td><td> AM9855G </td><td> pH值8.0 </td></tr><tr><td> 10X PBS </td><td> Ambion公司</td><td> AM9625 </td><td></td></tr><tr><td> 20X SSC的</td><td> Ambion公司</td><td> AM9763 </td><td></td></tr><tr><td> 1.5毫升microfuge管</td><td> Ambion公司</td><td> AM12400 </td><td>少不透明艾滋病可视化样品比标准管; </td></tr><tr><td>甲酰胺</td><td> Ambion公司</td><td> AM9342 </td><td>储存在4°C间</td></tr><tr><td> DEPC水</td><td> Ambion公司</td><td> AM9932 </td><td></td></tr><tr><td>蛋白酶K </td><td> Ambion公司</td><td> AM2546 </td><td></td></tr><tr><td> 5.25％次氯酸钠</td><td>奥斯汀</td><td></td><td>一个-1品牌</td></tr><tr><td> T3 RNA聚合酶加</td><td> Ambion公司</td><td> AM2733 </td><td>储存在-20°C </td></tr><tr><td> T7 RNA聚合酶</td><td> Ambion公司</td><td> AM2082 </td><td>储存在-20°C </td></tr><tr><td> 95％的乙醇</td><td> Fisher Scientific则</td><td> AC61511-0040 </td><td></td></tr><tr><td> fisherbrand一次性聚乙烯转移移液器</td><td> Fisher Scientific则</td><td> 13-711-7M </td><td></td></tr><tr><td> 37％的甲醛</td><td> Fisher Scientific则</td><td> F79-500 </td><td></td></tr><tr><td>高效液相色谱级甲醇</td><td> Fisher Scientific则</td><td> A452-1 </td><td></td></tr><tr><td>氯化镁</td><td> Fisher Scientific则</td><td>的M87-500 </td><td></td></tr><tr><td>显微镜盖玻片</td><td> Fisher Scientific则</td><td> 12-542A </td><td> 18×18毫米</td></tr><tr><td>正庚烷</td><td> Fisher Scientific则</td><td> H350-1 </td><td></td></tr><tr><td>对二甲苯</td><td> Fisher Scientific则</td><td> O5082-500 </td><td></td></tr><tr><td>耐热9点板</td><td> Fisher Scientific则</td><td> 13-748B </td><td> 100x85毫米</td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> AC32730-0025 </td><td></td></tr><tr><td>氢氧化钠</td><td> Fisher Scientific则</td><td> SS255-1 </td><td></td></tr><tr><td> superfrost / PLUS载玻片</td><td> Fisher Scientific则</td><td> 12-550-15 </td><td> 25x75X1.0毫米</td></tr><tr><td> davlyn红清除班轮发块磁带</td><td>头发直</td><td> RED-75R12 </td><td>聚/皮肤基料在0.75×12码胶带辊</td></tr><tr><td>一Sequening TOPOTA克隆试剂盒射击十佳化学主管大肠杆菌大肠杆菌 </td><td> Invitrogen公司</td><td> K457501 K457540 </td><td> 20个反应 40反应</td></tr><tr><td>超声波处理的鲑鱼精子DNA </td><td> Invitrogen公司</td><td> 15632-011 </td><td>储存在-20°C </td></tr><tr><td>抗地高辛-AP Fab片段</td><td>罗氏应用科学部</td><td> 1093274 </td><td>储存在4°C间</td></tr><tr><td>辛RNA的标签混合</td><td>罗氏应用科学部</td><td> 1277073 </td><td>储存在-20°C </td></tr><tr><td> NBT / BCIP原液</td><td>罗氏应用科学部</td><td> 1681451 </td><td>储存在4°C间</td></tr><tr><td>西方封锁试剂</td><td>罗氏应用科学部</td><td> 11921673001 </td><td>储存在4°C间</td></tr><tr><td> saatilene高新技术涤纶网眼（330.130） </td><td>纱帝打印</td><td> 330.34俄勒冈十五分</td><td> 330线程/英寸，螺纹直径34微米，橙色</td></tr><tr><td>甘油</td><td>西格玛</td><td> G6279-1 </td><td>在PBT的70％ </td></tr><tr><td>肝素钠盐</td><td>西格玛</td><td> H3393 </td><td></td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P1379-500 </td><td></td></tr><tr><td> 37％的甲醛</td><td>特德·佩拉</td><td> 18508 </td><td> 10毫升分装在琥珀色安瓿</td></tr><tr><td> 16盎司。有盖的单独纸容器</td><td>纸业公司</td><td> SOLOKH16AJ8000 </td><td></td></tr><tr><td>硼硅玻璃scintillat离子小瓶独立的螺丝帽</td><td> VWR国际</td><td> 66022-128 </td><td> 20毫升500例</td></tr><tr><td>密封的气泡包裹元胞衬垫材料</td><td> VWR国际</td><td> 500018-081 </td><td> 10英尺/辊0.188英寸厚</td></tr><tr><td>鸡血清</td><td></td><td></td><td>无论是从翼静脉或从少年鸡的心脏穿刺收集全血。在37°C孵育1小时，直到血液凝固，然后置于冰上30分钟。收集的血清离心10分钟在3000 XG。上清液（澄清血清）收集并储存在-20°C，直到使用。 </td></tr></tbody></table> 表4。表具体原位杂交试剂和设备。

hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes

蚊子是一套不同的病原体，包括虫媒病毒，原虫和线虫的媒介。在组织中的蚊子宿主和病原体相互作用，参与繁殖器官的极大兴趣战略，以减少蚊子传播的疾病传播和破坏卵的发育表达的转录和基因调控的研究。已经有许多工具时空和组织特异性基因表达调控的研究和验证。在这里，我们描述了已开发的协议，以获取空间信息，从而提高我们了解特定基因的表达和他们的产品积累。描述的协议已被用来验证的表达和蚊子传播的病原体传播有关的组织，如女性的唾液腺，以及卵巢和胚胎细胞内的车厢，确定誊积累模式，重新晚蚊子的繁殖和发展。 下列程序代表一个优化的方法，在协议中的各项步骤的效率提高没有特定目标的杂交信号的损失。 RNA探针的制备准则，软组织的解剖和一般的固定和杂交过程描述在A部分，B部分详细而具体步骤的收集，固定，预杂交和杂交蚊子胚胎中

Watch this Scientific Journal Video about Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes at JoVE.com

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

时间和空间上的基因表达分析在功能基因组学的重要作用。全挂载杂交<em&gt;原位</em&gt;是用于确定本地化组织和亚细胞车厢内的成绩单。在这里，我们勾勒1杂交<em&gt;原位</em&gt;蚊子的具体目标组织的修改协议。

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Research

JoVE Journal

Immunology and Infection

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ Hybridization on Mitotic Chromosomes 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. ...

A Method for the Measurement of Salivary Gland Function in Mice

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...