Name: visualization of microbiota in tick guts by whole-mount in situ hybridization
Uploaded: 2018-06-01T14:45:00.000+00:00
Duration: 8 min 30 s
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。 注意: 选择特定属属的基因区域是很重要的, 并确保所设计的引物不放大相关的细菌属。这是获得基因/属特异性探针杂交的关键。</li>
	<li>24, 48 或72小时的饲料蜱对小鼠, 解剖蜱内脏和准备 RNA 和 cDNA 从蜱内脏如前所述23。以 cDNA 为模板, 利用 thermocycler 对特异性 amplicons 进行 PCR 放大。在1.5% 琼脂糖凝胶上运行 PCR 产品/扩增子的整除, 并确认扩增子在紫外线 (UV) 光照射下使用凝胶成像系统与预期尺寸相对应。</li>
	<li>克隆细菌16S 核糖体 RNA 扩增子感兴趣的商业可用的 TA 向量(材料表) 包含 RNA 聚合酶促进剂适合噬菌体聚合酶 (SP6, T7 或 T3)。序列化克隆以确认扩增子的标识和克隆对每个启动子的方向性。在此基础上, 确定生成感知或反义 RNA 探针的选择的启动子 (图 1)。</li>
	<li>进行线性化1毫克质粒与适当的制约酵素在30µLfor 2 h 的反应容量在制造商推荐的温度。选择基于启动子的限制酶, 用于生成感觉或反义探针 (图 1)。验证2µL 1% 琼脂糖凝胶的消化反应的线性化。</li>
	<li>用一种商用的 PCR 产品柱纯化试剂盒提纯剩余的28µL, 用分光光度法定量测定 dna 浓度。 注意: 一些感测探头可能会导致背景染色远远高于相应的反义探针, 可能需要探索其他选择。替代性的阴性控制包括样品中没有表示的质粒编码序列或另一种能产生独特杂交模式的探针。</li>
</ol>2. Digoxygenin 双绞线 RNA 探针的构造
<ol><li>通过将7.5 µL 100 毫米 utp、25µL 10 毫米 digoxygenin 双绞线和10µL 每个100毫米的 ATP、GTP 和 CTP 在1.5 毫升离心管中组合, 制备核苷酸组合。</li>
	<li>使用1µL 的核苷酸组合 (步骤 2.1) 和 0.25-1 µg 模板 DNA, 利用商业上可用的 T7 或 Sp6 RNA 聚合酶套件执行体外转录。将体积高达20µL 水。轻拍管组合和脉冲旋转。孵育37°c 2.5-3 小时。 注: 探针结构已成功的消化质粒浓度低至7µL, 但典型的浓度在这个步骤范围从 15-25 ng/µL。转录反应也可能在一夜之间孵化37摄氏度, 但这并没有显著增加 RNA 的产量。</li>
	<li>添加1µL 的 DNase (2000 单位/µL) 和孵化37°c 10 分钟。不要超过10分钟, 否则酶就会开始降解 RNA。</li>
	<li>加入30µL 冰冷 7.5-8 米氯化锂和30µL 的冷冻核酸酶水, 以沉淀 RNA。轻弹管子混合。允许 RNA 的降雨雪在-20 °c 隔夜进行。</li>
	<li>以 1.7万 x g 为20分钟, 以4摄氏度的离心法收集 RNA。用1毫升新鲜制备的75% 乙醇在焦碳酸二乙酯 (DEPC) 水中一次清洗颗粒, 然后重复离心。</li>
	<li>除去并丢弃上清, 将管子倒在干净的纸巾上, 并允许干燥10分钟, 并用重悬在核酸酶水中的颗粒。如果颗粒很容易看到, 并用重悬在25µL。如果球团很小或不可见, 并用重悬在 15-20 µL. 通过分光光度法获得 RNA 浓度的估计。 注意: 典型的 RNA 浓度范围从 200-500 µL。</li>
</ol>3. rna 甲醛凝胶电泳 rna 探针的可视化评价 rna 纯度
注意: 甲醛构成吸入危险;因此, 在油烟机中生成 RNA 凝胶分析所需的解决方案。溴溴化物是一种疑似致癌物质;小心处理。
<ol><li>准备1% 甲醛琼脂糖凝胶如前所述的24 , 并将凝胶在一个无 RNases 的凝胶盒中。一旦冷却, 填充的凝胶盒与1x 运行缓冲器 (1x 拖把在 pH 值 7.0, 5% 甲醛)。</li>
	<li>准备样品缓冲器 (5 µL 400 毫克/毫升溴溴化物, 20 µL 10x 拖把, 35 µL 甲醛, 100 µL 甲酰胺)。将2µL 的 RNA 探针 (步骤 2.6) 与8µL 的样本缓冲器结合起来。孵育70°c 10 分钟。</li>
	<li>通过结合5毫升50% 甘油, 1 毫升10% 甲醛, 40 毫克溴酚蓝, 40 毫克二甲苯蓝, 20 µL EDTA (pH 0.5), 制备 RNA 加载缓冲器。使用后, 将此缓冲区存储在摄氏-20 摄氏度。</li>
	<li>从步骤3.2 和混合中添加3µL 的加载缓冲区到 RNA 样本。把样品管放在冰上。</li>
	<li>将整个采样卷从步骤3.4 加载到凝胶中。加载一个整除的单链 rna 梯子旁边, 以验证 RNA 的大小。用100伏或更低的凝胶, 直到蓝色染料迁移约75% 的方式下来的凝胶。使用凝胶成像系统在紫外线照射下对 RNA 进行可视化。 注意: 一个好质量的 RNA 探针应该显示为一个离散带, 具有与探测器的预期大小相对应的移动性 (图 2,车道 1)。如果探测器显示为漫射和油污波段 (图 2, 车道 2), 则不应使用此方法。</li>
</ol>4. 蜱肠收集和固定
<ol><li>收集Ixodes scapularis若虫, 它已经为小鼠喂食了感兴趣的时间点。 注: 为了这项技术的目的, 蜱喂养48或 72 h 最好的工作。</li>
	<li>在解剖显微镜下的玻璃滑梯上, 将肠道从蜱中解剖成 MEMFA (表 1), 尽可能避免对器官造成损伤。在 20-30 µL 的 MEMFA 上, 在干净的显微镜滑梯上放置一个刻度。使用一对不育的高碳钢剃刀刀片 #11 切片的头部区域盾牌座的蜱离开身体的蜱。轻轻按压身体, 将肠道憩和涎腺伸出身体角质层。把涎腺和内脏分开, 在剃刀刀片上舀出内脏。</li>
	<li>把内脏收集到一个1.5 毫升的管子里, 里面有500µL MEMFA。在室温下以1小时或一夜4摄氏度的温和摇摆的方法孵化试管。 注: 蜱腺也可解剖, 同样固定和染色。</li>
	<li>用1毫升冰冷100% 乙醇轻轻洗涤3次, 使组织脱水。让内脏在每次洗涤之间自然下沉到管子的底部, 因为离心可能会损伤组织。对于长期贮存, 请将样品在100% 乙醇中保持在-20 摄氏度。</li>
</ol>5. 网格样品篮和24井篮架的施工
<ol><li>用一把加热的单刃刀片在手术刀上切开2毫升或5毫升顶部离心管的底部。 注意: 刀片可能需要在切割完成前多次被加热。</li>
	<li>110µm 尼龙网格的切方形比新管开口稍大。将网格与管子的底部粘合在一起, 轻轻地压在中低热的热板上, 直到发出良好的密封。让冷却, 确保没有缝隙之间的网格和管, 并修剪多余的网格周围的边缘。</li>
	<li>在24井板的盖子上切开孔, 这样, 在步骤5.2 中制作的样品篮的唇会坐在洞里, 而不是掉下去, 产生一个设置, 在那里, 样品篮的底部将坐在井中所有的方式, 当他们放在盖子上有个洞。</li>
	<li>使用这个合适的篮子持有人把篮子从一个板块转移到另一个, 相对容易的整个就地杂交协议。使用两个24井板, 以便当一个孵化正在发生, 另一板块准备下一次清洗。</li>
</ol>6.原位杂交: 1 天 (时间: 4-5 h)
<ol><li>转移胆量到标记的样品篮子, 分离由实验情况和意欲的 RNA 探针。 注: 每个条件下染色至少5个内脏, 以解释复制之间的自然变化。</li>
	<li>水化, 通过逐步提高磷酸盐缓冲盐水与0.1% 非离子表面活性剂 (PTw) 的比例, 温和地避免引入气泡进入组织;表 1)在洗涤中的乙醇。 注: 在常温下用温和搅拌完成所有24井篮保持架的清洗, 除非另行注明。整除 500-600 µL 的洗涤液进入每一个井的持有人, 这样, 在每个篮子的胆量完全淹没。用 DEPC 处理的水制备所有溶液, 防止 RNA 降解。<ol>
<li>在500µL 100% 乙醇中清洗样品5分钟。</li>
		<li>准备至少500µL 每样篮子 75%, 50%, 25% 乙醇在 PTw. 水化样品通过洗涤每乙醇溶液中的5分钟, 从最高的乙醇浓度转移到最低。</li>
		<li>在 PTw 中每5分钟洗4次样品。</li>
	</ol>
</li>
	<li>用蛋白酶 K (10 µg/毫升) 在 PTw 中 Permeabilize 样品5分钟。</li>
	<li>准备与 RNA 探针杂交的组织。<ol>
<li>清洗样品两次, 如6.2 中所述24井篮持有人在常温下与温和搅拌5分钟每500µL 三乙醇胺缓冲 (0.1 米, pH 7.0-8.0) (表 1), 以保持在无胺环境中的样品。</li>
		<li>用三乙醇胺 (0.1 米, pH 7.0-8.0) 的500µL 0.25% 醋酸酐对样品进行两次处理, 分别为5分钟, 然后分别在 PTw 中清洗两次样品, 每次5分钟。 注: 醋酸酐 acetylates 游离胺中和正电荷, 这对于防止非特定静电相互作用和确保探针对 mRNA 的特异结合至关重要。</li>
		<li>固定的胆量为20分钟与500µL 4% 多聚甲醛在 PTw, 然后洗5次在 PTw 每5分钟。</li>
		<li>Prehybridize 通过孵化样品在500µL 杂交缓冲/在24井篮持有人在24井板 (表 1) 为 1.5-2.5 h 在60°c 与柔和的鼓动在摇晃的水浴。保存用于 prehybridization 步骤的杂交缓冲区, 以在2天协议 (步骤 7.1) 中重用。</li>
		<li>通过稀释杂交缓冲中的 RNA 探针, 制备探针杂交溶液, 最终浓度为1µg/毫升。在24井篮持有者的60°c 中用探针杂交解决方案孵化样品, 在摇晃的水浴中过夜 (表 2).  用铝箔紧贴着篮架的盘子, 以防止杂交溶液的蒸发。</li>
	</ol>
</li>
</ol>7.原位杂交: 2 天 (时间: 4.5-5.5 h)
<ol><li>从探针杂交解决方案中取出样本, 并将它们放入保留的杂交缓冲中, 从前一天开始。孵育60摄氏度, 温和搅拌3分钟。 注: 探针杂交解决方案可能存储在-20 摄氏度, 可重复使用多达3次。</li>
	<li>通过稀释 DEPC 处理水中的 20x SSC (表 1), 制备2x 盐水钠缓冲液 (SSC)。两次清洗样品3分钟与 2x ssc, 然后3倍20分钟与 2x SSC 在60°c, 以消除所有痕迹探针和杂交缓冲。</li>
	<li>以 RNase A (20 µg/毫升) 和 RNase T1 (10 µg/毫升) 为30分钟, 37 °c, 以除去代表自由非杂交 rna 的单链 rna 探针。</li>
	<li>室温下用 2x SSC 清洗一次, 10 分钟。通过稀释 DEPC 处理过的水中的 2x ssc 来制备 0.2x ssc, 然后用 0.2x ssc 清洗两次, 30 分钟到60摄氏度, 以去除多余的 RNases。</li>
	<li>用500µL 马来酸缓冲液清洗两次 (单克隆;表 1)每人10分钟。</li>
	<li>用阻断溶液孵化样品 (单克隆中的2% 个阻断剂), 1.5-2 小时。 注: 堵塞试剂不易溶解;温和加热有助于溶解。</li>
	<li>用抗辛碱性磷酸酶--共轭500µL 的抗体, 用1:3、000的稀释液在阻断溶液中代替阻塞溶液, 在室温下孵化约4小时或4摄氏度。</li>
</ol>8.原位杂交: 3 天 (时间: 6 小时到一夜之间)
<ol><li>用500µL 单克隆抗体对样品进行至少5次洗涤, 以去除过量抗血清。 注: 这些洗涤是灵活的, 可延长到 1-2 小时, 或 3-4 洗涤可能取代一夜间清洗在4°c, 对功效的影响极小。</li>
	<li>用碱性磷酸酶缓冲液清洗两次5分钟, pH 值 9.5 (AP 缓冲器;表 1)。</li>
	<li>先用500µL 的显色基底代替碱性磷酸酶 (材料表), 开始颜色反应。用铝箔覆盖样品板, 让染色在室温下进行 3-5 小时或一夜4摄氏度。在解剖显微镜下观察组织以避免 overstaining, 定期监测染色反应。 注意: 当染色完成后, 在显微镜下反义探测样品中, 可以用眼睛检测到紫色的色调, 但是组织不应被允许变得非常暗。染色的进展非常缓慢, 在4摄氏度, 可能需要多达 20-24 小时。</li>
	<li>通过在500µL 单克隆中洗涤5分钟来停止颜色反应, 然后在 Bouin 的溶液中过夜修复组织。 注意: 在通风罩中处理 Bouin 的溶液;这是一种腐蚀性致癌物质。</li>
</ol>9.原位杂交: 4 天 (时间: 3-3.5 h)
<ol><li>在 DEPC 处理的水中稀释 20x ssc, 每样 1x ssc 的样品篮中至少准备7毫升。在 1x SSC 中用70% 乙醇洗涤样品4到6倍, 直到组织不再是黄色时, 去掉 Bouin 的溶液。</li>
	<li>在 1x SSC 中, 每样篮子准备500µL 75%、50% 和25% 乙醇溶液。在每个溶液中清洗样品5分钟, 从最高乙醇浓度到最低水化组织。在 1x SSC 中每两次清洗5分钟。</li>
	<li>将漂白液中的样品 (表 1) 孵化为通风罩中灯箱上的90分钟。 注意: 这一步允许样品中的任何色素沉着或从 Bouins 溶液中着色, 并增强探针信号, 以清晰可视化。</li>
	<li>用500µL 1x SSC 清洗样品两次, 5 分钟。</li>
	<li>通过将样品篮反转到一个新的24井板中, 然后用转移吸管在网底上冲洗70% 乙醇, 以最小的损伤来迁移内脏。如果需要, 存储在-20 摄氏度。</li>
	<li>要获得对多个样本的染色模式的概述, 如图 3所示,在24井板中保持70% 乙醇中的内脏, 并与任何明亮的场显微镜进行观察。要检查明亮场显微镜下的单个内脏, 如图 4、图 5和图 6所示, 请在盖玻片下的100% 甘油中安装内脏。使用塑料铲轻轻地操纵组织与最小的伤害。 注: 高粘度甘油有助于保护组织免受压迫, 并允许仔细定位憩, 使组织可以在更高的放大的最佳查看。</li>
	<li>使用显微镜特定的图像捕获软件 (材料表) 在显微镜上捕获图像, 并将其作为 Tiff 图像导出到通用图像编辑软件中。为了量化实验治疗之间的相对染色差异, 下载一个通用图像分析软件 (材料表)。在分析软件中打开图像, 并使用软件中的指令测量染色的像素强度, 并计算染色的直方图图。</li>
</ol>

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作者没有披露的利益冲突。

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。

<table><tbody><tr><td>Sefar NITEX Nylon Mesh, 110 micron</td><td>Amazon</td><td>03-110/47</td><td></td></tr><tr><td>pGEM-T Easy Vector System</td><td>Promega</td><td>A1360</td><td></td></tr><tr><td>Digoxygenin-11-UTP</td><td>Roche</td><td>1209256910</td><td></td></tr><tr><td>dNTP</td><td>New England Biolabs&amp;nbsp;</td><td>N0447S</td><td></td></tr><tr><td>DNAse I(RNAse-free)</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>HiScribe SP6 RNA synthesis kit</td><td>New England Biolabs</td><td>E2070S</td><td></td></tr><tr><td>HiScribe T7 High Yield RNA Synthesis Kit</td><td>New England Biolabs</td><td>E2040S</td><td></td></tr><tr><td>Water, RNase-free, DEPC-treated</td><td>American Bioanalytical</td><td>AB02128-00500</td><td></td></tr><tr><td>EDTA, 0.5M, pH 8.0</td><td>American Bioanalytical</td><td>AB00502-01000</td><td></td></tr><tr><td>Formaldehyde, 37%</td><td>JT Baker</td><td>2106-01</td><td></td></tr><tr><td>Formamide</td><td>American Bioanalytical</td><td>AB00600-00500</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E-4378</td><td></td></tr><tr><td>DPBS, 10X</td><td>Gibco</td><td>14300-075</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379-25ML</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>3115879001</td><td></td></tr><tr><td>Triethanolamine HCl</td><td>Sigma Aldrich</td><td>T1502-100G</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma Aldrich</td><td>320102-100ML</td><td></td></tr><tr><td>Paraformaldehyde</td><td>ThermoScientific/Pierce</td><td>28906</td><td></td></tr><tr><td>SSC, 20X</td><td>American Bioanalytical</td><td>AB13156-01000</td><td></td></tr><tr><td>RNA from torula yeast</td><td>Sigma Aldrich</td><td>R3629-5G</td><td></td></tr><tr><td>Heparin, sodium salt</td><td>Sigma Aldrich</td><td>H3393-10KU</td><td></td></tr><tr><td>Denhardt&#039;s Solution, 50X</td><td>Sigma Aldrich</td><td>D2532-5ML</td><td></td></tr><tr><td>CHAPS hydrate</td><td>Sigma Aldrich</td><td>C3023-1G</td><td></td></tr><tr><td>RNase A</td><td>Sigma Aldrich</td><td>10109142001</td><td></td></tr><tr><td>RNase T1</td><td>ThermoScientific&amp;nbsp;</td><td>EN0541</td><td></td></tr><tr><td>Maleic acid</td><td>Sigma Aldrich</td><td>M0375-100G</td><td></td></tr><tr><td>Blocking reagent</td><td>Sigma Aldrich</td><td>11096176001</td><td></td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Sigma Aldrich</td><td>11093274910</td><td></td></tr><tr><td>Levamisol hydrochloride</td><td>Sigma Aldrich</td><td>31742-250MG</td><td></td></tr><tr><td>Chromogenic substrate for alkaline phosphatase</td><td>Sigma Aldrich</td><td>11442074001</td><td></td></tr><tr><td>Bouin&#039;s solution</td><td>Sigma Aldrich</td><td>HT10132-1L</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Mallinkrodt Baker, Inc</td><td>2186-01</td><td></td></tr><tr><td>Single stranded RNA ladder</td><td>Ambion -Millenium</td><td>AM7151</td><td></td></tr><tr><td>&amp;nbsp;#11 High-Carbon steel blades&amp;nbsp;</td><td>C and A Scientific Premiere</td><td>#11-9411</td><td></td></tr><tr><td>Thermocycler</td><td>BioRad, CA</td><td>1851148</td><td></td></tr><tr><td>Spectrophotometer</td><td>ThermoScientific&amp;nbsp;</td><td>NanoDrop 2000C</td><td></td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>DS-500E Digital Orbital shaker</td><td></td></tr><tr><td>Shaking water bath</td><td>BELLCO Glass, Inc</td><td>Hot Shaker-7746-12110</td><td></td></tr><tr><td>Gel&amp;nbsp; documentation system</td><td>BioRad</td><td>Gel Doc XR+ Gel documentation system</td><td></td></tr><tr><td>Bright-field Microscope&amp;nbsp;</td><td>Nikon</td><td>NikonSM2745T</td><td></td></tr><tr><td>Bright-field Microscope&amp;nbsp;</td><td>Zeiss</td><td>AXIO Scope.A1</td><td></td></tr><tr><td>Dissection microscope&amp;nbsp;</td><td>Zeiss</td><td>STEMI 2000-C</td><td></td></tr><tr><td>&amp;nbsp;Light box</td><td>VWR</td><td>102097-658</td><td></td></tr><tr><td>PCR purification kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Image capture software</td><td>Zeiss</td><td>Zen lite</td><td></td></tr><tr><td>Image editing software&amp;nbsp;</td><td>Adobe&amp;nbsp;</td><td>Adobe Photoshop CS4 version 11.0</td><td></td></tr><tr><td>Image analysis software</td><td>National Institutes of Health</td><td>ImageJ-NIH /imagej.nih.gov/ij/</td><td></td></tr><tr><td>Automation compatible instrumentation</td><td>Intavis Bioanalytical Instruments, Tubingen, Germany).&amp;nbsp;</td><td>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 ...

visualization-microbiota-tick-guts-whole-mount-situ

Research

JoVE Journal

Genetics

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

9.5K 次观看.  Yale University School of Medicine. 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。

视频: 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

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

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

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

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

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

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

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

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

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

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 ...

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

免疫与感染

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

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 ...

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 ...

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦:揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

全挂载原位杂交法可视化蜱内脏微生物群

摘要

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通过多色荧光跨染色体畸变稳定的检测

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

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

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

全挂载原位杂交法可视化蜱内脏微生物群

摘要

探索更多视频

此视频中的章节

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

光遗传学随机突变用组蛋白miniSOG在

通过多色荧光跨染色体畸变稳定的检测

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

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