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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	<li>Juhn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+mapping+of+gene+expression+in+the+salivary+glands+of+the+dengue+vector+mosquito,+Aedes+aegypti.">Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, Aedes aegypti.</a> Parasit. Vectors. 4, 1 (2011).</li><li>Coleman, J., Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+midgut+and+salivary+glands+from+Ae.+aegypti+mosquitoes.">Dissection of midgut and salivary glands from Ae. aegypti mosquitoes.</a> J. Vis. Exp. (5), e228 (2007).</li><li>Benedict, M. 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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+Embryo+Fixation,+Chapter.+3.7.">Anopheles Embryo Fixation, Chapter. 3.7.</a> Methods in Anopheles Research. Malaria Research and Reference Reagent Resource Center (MR4). , (2007).</li><li>Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=oskar+gene+expression+in+the+vector+mosquitoes,+Anopheles+gambiae+and+Aedes+aegypti.">oskar gene expression in the vector mosquitoes, Anopheles gambiae and Aedes aegypti.</a> Insect Mol. Biol. 15, 363-372 (2006).</li><li>Juhn, J., Marinotti, O., Calvo, E., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+structure+and+expression+of+nanos+(nos)+and+oskar+(osk)+orthologues+of+the+vector+mosquito,+Culex+quinquefasciatus.">Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus.</a> Insect Mol. Biol. 17, 545-552 (2008).</li><li>Raminani, L. N., Cupp, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Embryology+of+Aedes+aegypti+(L.)+(Diptera:+Culicidae).">Early Embryology of Aedes aegypti (L.) (Diptera: Culicidae).</a> Int. J. Insect Morphol. &#38; Embryol. 4, 517-528 (1975).</li><li>Raminani, L. N., Cupp, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryology+of+Aedes+aegypti+(L.)+(Diptera:+Culicidae):+Organogenesis.">Embryology of Aedes aegypti (L.) (Diptera: Culicidae): Organogenesis.</a> Int. J. Insect morphol. &#38; Embryol. 7, 273-296 (1978).</li><li>Ivanova-Kazas, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+development+of+Anopheles+maculipennis+Mg.">Embryonic development of Anopheles maculipennis Mg.</a> Izv. Akad. Nauk SSSR ser. Biol. 2, 140-170 (1949).</li><li>Goltsev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+evolutionary+basis+for+drought+tolerance+of+the+Anopheles+gambiae+embryo.">Developmental and evolutionary basis for drought tolerance of the Anopheles gambiae embryo.</a> Dev. Biol. 330, 462-470 (2009).</li><li>Papatsenko, D., Levine, M., Goltsev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clusters+of+temporal+discordances+reveal+distinct+embryonic+patterning+mechanisms+in+Drosophila+and+Anopheles.">Clusters of temporal discordances reveal distinct embryonic patterning mechanisms in Drosophila and Anopheles.</a> PLoS Biol. 9, e1000584 (2011).</li><li>Davis, C. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+larval+embryogenesis+in+the+mosquito+Culex+fatigans+Wiedemann+(Diptera:+Culicidae)+and+the+sheep-fly+Lucilla+seriata+Meigen+(Diptera:+Calliphoridae)+I.+Description+of+embryonic+development.">A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilla seriata Meigen (Diptera: Calliphoridae) I. Description of embryonic development.</a> Aust. J. Zool. 15, 547-579 (1967).</li></ol>

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 ...

hybridization-situ-salivary-glands-ovaries-embryos-vector

Research

JoVE Journal

Immunology and Infection

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

16.4K 次观看.  University of California, Irvine. 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。全挂载杂交原位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杂交原位蚊子的具体目标组织的修改协议。

视频: 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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

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

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

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

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

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

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

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 (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. ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

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

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

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

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

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

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

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

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 colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

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

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

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

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

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

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

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 ...