Name: visualization of microbiota in tick guts by whole-mount in situ hybridization
Uploaded: 2018-06-01T14:45:00
Description: Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

我们衷心感谢耶鲁大学的穆斯塔法 Khokha 博士提供了他的实验室资源的使用。我们感谢明洁吴先生的出色技术援助。EF 是 HHMI 的调查员。这项工作得到了来自约翰 Monsky 和珍妮弗毛巾 Monsky 莱姆疾病研究基金的礼物的支持。

1. DNA 模板的制备
<ol><li>从 NCBI 数据库中下载特定于每个细菌属的 16S rRNA 序列, 并设计聚合酶链反应 (PCR) 相容的正向和反向引物, 将放大特定区域 (~ 200-250 基对长), 如前所述19. 还请参阅开源数据库席尔瓦20、21和 probeBase22在线资源, 用于 rRNA 目标的寡核苷酸探针和引物, 因为某些细菌属的 RNA 探针可能是商业性的。可用...

<ol>
	<li>Leitner, W. W., Wali, T., Kincaid, R., Costero-Saint Denis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthropod+Vectors+and+Disease+Transmission:+Translational+Aspects.">Arthropod Vectors and Disease Transmission: Translational Aspects.</a> PLoS Neglected Tropical Diseases. 9 (11), e0004107 (2015).</li><li>Hooper, L. V., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commensal+host-bacterial+relationships+in+the+gut.">Commensal host-bacterial relationships in the gut.</a> Science. 292 (5519), 1115-1118 (2001).</li><li>Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worlds+within+worlds:+evolution+of+the+vertebrate+gut+microbiota.">Worlds within worlds: evolution of the vertebrate gut microbiota.</a> Nature Review Microbiology. 6 (10), 776-788 (2008).</li><li>Narasimhan, S., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+microbiome:+the+force+within.">Tick microbiome: the force within.</a> Trends in Parasitology. 31 (7), 315-323 (2015).</li><li>Weiss, B., Aksoy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiome+influences+on+insect+host+vector+competence.">Microbiome influences on insect host vector competence.</a> Trends in Parasitoly. 27 (11), 514-522 (2011).</li><li>Degli Esposti, M., Martinez Romero, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+microbiome+of+arthropods.">The functional microbiome of arthropods.</a> PLoS One. 12 (5), e0176573 (2017).</li><li>Radolf, J. D., Caimano, M. J., Stevenson, B., Hu, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+ticks,+mice+and+men:+understanding+the+dual-host+lifestyle+of+Lyme+disease+spirochaetes.">Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.</a> Nature Reviews Microbiology. 10 (2), 87-99 (2012).</li><li>Sojka, 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=New+insights+into+the+machinery+of+blood+digestion+by+ticks.">New insights into the machinery of blood digestion by ticks.</a> Trends in Parasitology. 29 (6), 276-285 (2013).</li><li>Grigor'eva, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphofunctional+changes+in+the+midgut+of+Ixodid+ticks+during+the+life+cycle.">Morphofunctional changes in the midgut of Ixodid ticks during the life cycle.</a> Entomological Review. 90, 405-409 (2010).</li><li>Goodrich, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+a+microbiome+study.">Conducting a microbiome study.</a> Cell. 158 (2), 250-262 (2014).</li><li>Salter, S. J., 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=Reagent+and+laboratory+contamination+can+critically+impact+sequence-based+microbiome+analyses.">Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.</a> BMC Biology. 12, 87 (2014).</li><li>Hemmati-Brivanlou, A., 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=Localization+of+specific+mRNAs+in+Xenopus+embryos+by+whole-mount+in+situ+hybridization.">Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.</a> Development. 110 (2), 325-330 (1990).</li><li>Frank, D., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+of+XMyoD+in+non-somitic+mesoderm+of+Xenopus+gastrulae.">Transient expression of XMyoD in non-somitic mesoderm of Xenopus gastrulae.</a> Development. 113 (4), 1387-1393 (1991).</li><li>Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+an+improved+whole-mount+method+for+Xenopus+embryos.">In situ hybridization: an improved whole-mount method for Xenopus embryos.</a> Methods in Cell Biology. 36, 685-695 (1991).</li><li>Kernohan, K. D., Berube, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dimensional+dual+labelled+DNA+fluorescent+in+situ+hybridization+analysis+in+fixed+tissue+sections.">Three dimensional dual labelled DNA fluorescent in situ hybridization analysis in fixed tissue sections.</a> MethodsX. 1, 30-35 (2014).</li><li>Sonenshine, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of ticks. , (1991).</li><li>Narasimhan, 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=Gut+microbiota+of+the+tick+vector+Ixodes+scapularis+modulate+colonization+of+the+lyme+disease+spirochete.">Gut microbiota of the tick vector Ixodes scapularis modulate colonization of the lyme disease spirochete.</a> Cell Host Microbe. 15 (1), 58-71 (2014).</li><li>Hammer, B., Moter, A., Kahl, O., Alberti, G., Gobel, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Borrelia+burgdorferi+sensu+lato+by+fluorescence+in+situ+hybridization+(FISH)+on+whole-body+sections+of+Ixodes+ricinus+ticks+and+gerbil+skin+biopsies.">Visualization of Borrelia burgdorferi sensu lato by fluorescence in situ hybridization (FISH) on whole-body sections of Ixodes ricinus ticks and gerbil skin biopsies.</a> Microbiology. 147 (Pt 6), 1425-1436 (2001).</li><li>Harmsen, H. J., Raangs, G. C., He, T., Degener, J. E., Welling, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+set+of+16S+rRNA-based+probes+for+detection+of+bacteria+in+human+feces.">Extensive set of 16S rRNA-based probes for detection of bacteria in human feces.</a> Applied Environmental Microbiology. 68 (6), 2982-2990 (2002).</li><li>Quast, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SILVA+ribosomal+RNA+gene+database+project:+improved+data+processing+and+web-based+tools.">The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.</a> Nucleic Acids Research. 41 (Database issue), D590-D596 (2013).</li><li>Yilmaz, 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=The+SILVA+and+&amp;#34;All-species+Living+Tree+Project+(LTP)&amp;#34;+taxonomic+frameworks.">The SILVA and &amp;#34;All-species Living Tree Project (LTP)&amp;#34; taxonomic frameworks.</a> Nucleic Acids Research. 42, D643-D648 (2014).</li><li>Greuter, D., Loy, A., Horn, M., Rattei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probeBase--an+online+resource+for+rRNA-targeted+oligonucleotide+probes+and+primers:+new+features+2016.">probeBase--an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.</a> Nucleic Acids Research. 44, D586-D589 (2016).</li><li>Narasimhan, 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=Modulation+of+the+tick+gut+milieu+by+a+secreted+tick+protein+favors+Borrelia+burgdorferi+colonization.">Modulation of the tick gut milieu by a secreted tick protein favors Borrelia burgdorferi colonization.</a> Nature Communication. 8 (1), 184 (2017).</li><li>Bryant, S., Manning, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+gel+electrophoresis+of+total+RNA.">Formaldehyde gel electrophoresis of total RNA.</a> Methods Molecular Bioliogy. 86, 69-72 (1998).</li><li>Tautz, D., Pfeifle, 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+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98 (2), 81-85 (1989).</li><li>Robson, A., Owens, N. D., Baserga, S. J., Khokha, M. K., Griffin, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+ribosomopathy+genes+during+Xenopus+tropicalis+embryogenesis.">Expression of ribosomopathy genes during Xenopus tropicalis embryogenesis.</a> BMC Development Biology. 16 (1), 38 (2016).</li><li>Khokha, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+probes+for+the+study+of+Xenopus+tropicalis+development.">Techniques and probes for the study of Xenopus tropicalis development.</a> Developmental Dynamics. 225 (4), 499-510 (2002).</li><li>Jowett, T., Lettice, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridizations+on+zebrafish+embryos+using+a+mixture+of+digoxigenin-+and+fluorescein-labelled+probes.">Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes.</a> Trends in Genetics. 10 (3), 73-74 (1994).</li><li>Behnam, F., Vilcinskas, A., Wagner, M., Stoecker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+straightforward+DOPE+(double+labeling+of+oligonucleotide+probes)-FISH+(fluorescence+in+situ+hybridization)+method+for+simultaneous+multicolor+detection+of+six+microbial+populations.">A straightforward DOPE (double labeling of oligonucleotide probes)-FISH (fluorescence in situ hybridization) method for simultaneous multicolor detection of six microbial populations.</a> Applied Environmental Microbiology. 78 (15), 5138-5142 (2012).</li><li>Pai, V. 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=HCN4+ion+channel+function+is+required+for+early+events+that+regulate+anatomical+left-right+patterning+in+a+nodal+and+lefty+asymmetric+gene+expression-independent+manner.">HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner.</a> Biology Open. 6 (10), 1445-1457 (2017).</li><li>Legendre, F., 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=Whole+mount+RNA+fluorescent+in+situ+hybridization+of+Drosophila+embryos.">Whole mount RNA fluorescent in situ hybridization of Drosophila embryos.</a> JoVE. (71), e50057 (2013).</li></ol>

这是首次使用全装入式原位杂交 (WMISH) 技术研究病原体节肢动物载体的微生物群。我们的协议是从一个用于研究开发在果蝇和青蛙胚胎25,26。全挂载 RNA原位杂交已被常规地用于本地化基因转录的空间和世俗的27和可视化的转录可以做的明亮领域或荧光显微镜。我们采用前者对蜱内脏的微生物进行检测。需要注意的是...

节肢动物传染媒介传播的人类和家畜病原体遍布全球, 占全球20% 的传染性疾病的1, 但对大多数这些病原体没有有效和安全的疫苗可供使用。我们对共生、共生和致病微生物的重要作用的理解, 统称为微生物群2, 在调制和塑造几乎所有后生3的健康方面都在扩大。现在很明显, 病原体的节肢动物载体也窝藏肠道微生物群, 这些与载体相关的微生物群落已经被证明对不同的载体传染病原体有影响4,5。节肢动物微生物群由 eubacteria、古菌、病毒和真核微生物组成, 如原生动物、线虫和真菌6。然而, 主要的研究重点是 eubacteria, 部分原因是可获得的标记基因和参考数据库, 以确定特定的细菌成员。
以Ixodes scapularis为重点, 包括螺旋体螺旋体7、病致病剂等多种人类病原体的蜱向量, 对一种微生物可视化技术进行了优化, 旨在在媒介-病原菌相互作用的背景下, 提高我们对蜱肠道微生物群的认识。在蜱微生物场中仍有几个问题有待回答。肠道是在水平转移病原体背景下, 蜱与传入病原体之间第一次延长相遇的地点;因此, 了解载体肠道微生物群在调节载体-病原体相互作用中的作用将揭示有意义的见解。蜱有一个独特的模式的血液膳食消化, 其中处理的血液膳食成分发生 intracellularly8。肠道腔似乎作为一个容器, 以控制血液的膳食作为蜱饲料, 和养分消化和同化在数天的喂养和继续后饱食。在喂食过程中获得的病原体与 bloodmeal 一起进入肠腔, 因此流明成为蜱、病原体和居民微生物间相互作用的主要部位。随着消化通过饱食和Ixodid蜱蜕皮, 肠道经历结构和功能的变化9。肠道细菌的组成和空间组织也可能随着肠道环境的变化而变化。因此, 重要的是要了解蜱肠道内细菌的结构, 以充分了解蜱、病原体和肠道微生物群的相互作用。
描述宿主相关微生物群的分子技术经常利用高通量并行排序策略10来放大和序列细菌16S 核糖体 DNA (rDNA)。这些排序策略规避了获取特定细菌的无菌文化的需要, 并对样本中所代表的所有细菌成员提供了深入的描述。然而, 这种战略因无法区分环境污染物与善意居民而混淆。此外, 在评估样本 (如蜱) 时, 体积小, 因而含有低菌群特定的 DNA 产量时, 环境污染物放大的可能性增加了11 , 并导致模糊的解释微生物组成。因此, 与特定生存细菌的可视化相结合的功能表征, 对于确定和辨别蜱虫菌群在世俗和空间上都是至关重要的。为了实现这个目标, 我们利用了全安装 RNA原位杂交。这种技术通常用于评估器官和胚胎中的基因表达模式12,13,14 , 并允许定量分析整个样本的兴趣。这不同于传统的就地杂交技术, 利用组织切片, 通常需要对分段材料进行广泛的分析, 用一个计算组件来预测整个器官中的表达式15。当整体装载一般提到整个有机体12, 这里整体装载提到整体内脏或器官。采用全挂载 RNA原位杂交方法评价蜱肠菌群的体系结构的优点多种。蜱肠道由7对憩组成, 每对变化大小为16。功能差异, 如果有的话, 在这些憩, 是不理解的背景下蜱生物学, 蜱微生物群或蜱病原体相互作用。肠道憩的操作将会取代肠道腔内的微生物群, 或与肠道松散相关的人, 导致对微生物的空间定位产生误解。使用荧光标记的 RNA原位杂交在早期通过固定和打开个体肠道憩来检查蜱肠记录17 , 以确保探针杂交和本地化B. 螺旋体记录通过切片石蜡嵌入的整个刻度18。这两种方法都需要在杂交之前对蜱组织进行操作, 这会影响肠道微生物体系结构。
在本报告中, 我们详细描述了使用全装入式原位杂交 (WMISH) 检查可行蜱肠道微生物群的协议。使用全挂载 RNA原位杂交使全球了解肠道不同区域内特定肠道细菌的存在和丰度, 并可能在病原体殖民化的背景下激发对蜱肠生物学的新见解。和传输。此外, 使用 rna 探针针对特定的细菌 rna, 可以检测细菌在蜱肠道。

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			<td height="21" style="height:21px;width:320px;">Sefar NITEX Nylon Mesh, 110 micron</td>
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			<td height="21" style="height:21px;width:320px;">pGEM-T Easy Vector System</td>
			<td style="width:220px;">Promega</td>
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			<td></td>
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			<td height="21" style="height:21px;width:320px;">Digoxygenin-11-UTP</td>
			<td style="width:220px;">Roche</td>
			<td align="right" style="width:348px;">1209256910</td>
			<td></td>
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			<td height="21" style="height:21px;width:320px;">dNTP</td>
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			<td height="21" style="height:21px;width:320px;">DNAse I(RNAse-free)</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe SP6 RNA synthesis kit</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe T7 High Yield RNA Synthesis Kit</td>
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			<td height="21" style="height:21px;width:320px;">Water, RNase-free, DEPC-treated</td>
			<td style="width:220px;">American Bioanalytical</td>
			<td style="width:348px;">AB02128-00500</td>
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			<td height="21" style="height:21px;width:320px;">EDTA, 0.5M, pH 8.0</td>
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			<td height="21" style="height:21px;width:320px;">Formaldehyde, 37%</td>
			<td style="width:220px;">JT Baker</td>
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			<td height="21" style="height:21px;width:320px;">Formamide</td>
			<td style="width:220px;">American Bioanalytical</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EGTA</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">E-4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">DPBS, 10X</td>
			<td style="width:220px;">Gibco</td>
			<td style="width:348px;">14300-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tween-20</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">P1379-25ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Proteinase K</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Triethanolamine HCl</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">T1502-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Acetic anhydride</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">320102-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Paraformaldehyde</td>
			<td style="width:220px;">ThermoScientific/Pierce</td>
			<td align="right" style="width:348px;">28906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">SSC, 20X</td>
			<td style="width:220px;">American Bioanalytical</td>
			<td style="width:348px;">AB13156-01000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RNA from torula yeast</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">R3629-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Heparin, sodium salt</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">H3393-10KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Denhardt&#39;s Solution, 50X</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">D2532-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">CHAPS hydrate</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">C3023-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RNase A</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">10109142001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RNase T1</td>
			<td style="width:220px;">ThermoScientific&#160;</td>
			<td style="width:348px;">EN0541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Maleic acid</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">M0375-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Blocking reagent</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-Digoxigenin-AP, Fab fragments</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Levamisol hydrochloride</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">31742-250MG</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:320px;">Chromogenic substrate for alkaline phosphatase</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11442074001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bouin&#39;s solution</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">HT10132-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hydrogen peroxide</td>
			<td style="width:220px;">Mallinkrodt Baker, Inc</td>
			<td style="width:348px;">2186-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Single stranded RNA ladder</td>
			<td style="width:220px;">Ambion -Millenium</td>
			<td style="width:348px;">AM7151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;#11 High-Carbon steel blades&#160;</td>
			<td style="width:220px;">C and A Scientific Premiere</td>
			<td style="width:348px;">#11-9411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Thermocycler</td>
			<td style="width:220px;">BioRad, CA</td>
			<td align="right" style="width:348px;">1851148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Spectrophotometer</td>
			<td style="width:220px;">ThermoScientific&#160;</td>
			<td style="width:348px;">NanoDrop 2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Orbital shaker</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">DS-500E Digital Orbital shaker</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Shaking water bath</td>
			<td style="width:220px;">BELLCO Glass, Inc</td>
			<td style="width:348px;">Hot Shaker-7746-12110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Gel&#160; documentation system</td>
			<td style="width:220px;">BioRad</td>
			<td style="width:348px;">Gel Doc XR+ Gel documentation system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Nikon</td>
			<td style="width:348px;">NikonSM2745T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">AXIO Scope.A1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dissection microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">STEMI 2000-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Light box</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">102097-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PCR purification kit&#160;</td>
			<td style="width:220px;">Qiagen</td>
			<td align="right" style="width:348px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image capture software</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">Zen lite</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image editing software&#160;</td>
			<td style="width:220px;">Adobe&#160;</td>
			<td style="width:348px;">Adobe Photoshop CS4 version 11.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image analysis software</td>
			<td style="width:220px;">National Institutes of Health</td>
			<td>ImageJ-NIH /imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;">Automation compatible instrumentation</td>
			<td style="width:220px;">Intavis Bioanalytical Instruments, Tubingen, Germany).&#160;</td>
			<td style="width:348px;">Intavis, Biolane HT1.16v</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of microbiota in tick guts by whole-mount in situ hybridization

节肢动物传染媒介传播的传染病继续对全世界的人类健康构成严重威胁。引起这些疾病的病原体, 在殖民载体时不存在孤立;相反, 它们很可能与肠道内的微生物发生相互作用。载体菌群在病原体传播中起着重要的作用。是否细菌在肠道内的Ixodes scapularis蜱, 多人病原体的载体, 包括螺旋体螺旋体,影响蜱传播的病原体是不确定的。我们需要方法来表征与蜱肠相关的细菌组成, 以促进更好地了解在蜱肠道内的潜在的种间相互作用。使用全装入的就地杂交可视化与特定细菌种类相关的 RNA 记录, 可以收集有关完整组织中微生物的丰度和分布的定性数据。该技术可用于检测蜱喂养过程中肠道微生物群落环境的变化, 也可用于分析蜱基因的表达。全蜱内脏的染色在不需要三维重建的情况下, 对组织中目标 RNA 的总空间分布进行信息分析, 对环境污染的影响较小, 往往会混淆基于排序的方法经常用于研究复杂的微生物群落。总的来说, 这项技术是一个有价值的工具, 可以用来更好地了解病原体-微生物群的相互作用及其在疾病传播中的作用。

RNA 探针质量的测量和估计在开始染色之前是关键的。体外转录效率高度取决于 DNA 模板的数量和质量。我们经常在甲醛凝胶上可视化 RNA 探针, 以验证转录反应产生的探针的纯度和数量。探测器应显示为明亮的、离散的波段 (图 2)。分光光度法测定 RNA 浓度, 对探针质量有很高的指示性, 与凝胶带的外观有很好的相关性。因此, 一旦熟悉了协议, 就...

Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

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

全挂载原位杂交法可视化蜱内脏微生物群

在这里, 我们提出一个协议, 以空间和世俗评估的存在, 在蜱内脏有活力的微生物, 使用改良的全芒原位杂交方法。

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

Research

JoVE Journal

Genetics

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

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

提高墨西哥龙舌兰对幼虫后表型和全体免疫组织化学的分析

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...