Name: a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas
Uploaded: 2014-06-05T14:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

作者要感谢以下美国国立卫生研究院奖项/ subawards对本研究的支持:R01HL106790，R01HL106790-S1，R01HL106786，R01AI089323，R21AI091457，R21RR026006，P20RR020159，C06RR017563，8T32OD011124-08，和P51OD011104。

1，校准Mesodissection仪器与2ID成像软件该2ID成像软件将被称为无论是软件或程序为本文的其余部分。这一步是需要正确地跟踪图像以及对齐多个幻灯片框架。 <ol><li>打开仪器和计算机。 </li><li>打开软件，选择"校准仪器"。 </li><li>通过使用操纵杆校准阶段行程。移动舞台左上角的位置。按"设置"下的程序步骤1。 </li><li>现阶段移动到右下角的位置。按"设置"下的程序步骤2。 </li><li>首先按"Z键"，以提高磁头组件校准愿望。按"吸脉冲"按钮和"解剖"按钮操纵杆，直到柱塞控制杆到达顶部位置。按"设置"下的程序的步骤3。 </li><li>按下"抽吸复位＆＃8221;按钮。一旦活塞停止在步骤4中的底部位置按"SET"。 </li><li>点击"刻度标签"上。 </li><li>将校准标尺上的空白一面。 </li><li>如果需要集中显微镜。 </li><li>绘制鼠标使​​用两比例尺之间的线。 </li><li>在标第2步，按"计算"按钮，进入距离。 </li><li>点击"十字准线"选项卡。转电机转速为1。 </li><li>将xScisor成头组件。下磁头组装成z轴的准备位置，按"z轴"按钮。注:头组件在顶部的解剖仪器的大型结构。该xScisor将被称为耗材厂位在本文的其余部分。 </li><li>点击"解剖搞"按钮操纵杆。注:此按钮位于操纵杆顶部。 </li><li>定义旋转中心视觉。要做到这一点，使用定义移动十字线的中心在旋转中心在程序的步骤1 d参数。使用向上和向下箭头在"x"和"y轴"条来调整像素对齐的十字线。点击"完成"选项卡。 </li><li>点击"主页"标志。注意:这是由位于右上角的房子形象代表。 </li></ol> 2，创建参考图像<ol><li>将H＆E幻灯片上舞台。将不透明的盖子顶部的滑动。通过移动操纵杆H＆E幻灯片的所需区域带动仪器。保存生成的图像。注意:无论是"捕获引用"选项卡或"解剖"选项卡可以用来完成此步骤。 </li></ol> 3，对准组织的解剖<ol><li>点击主屏幕上的"解剖组织"选项。 </li><li>填写"经营者"，"解剖登录号"，"参考入藏号"，"xScisor条码号，"＆＃8220;剥离液批号"，"xScisor大小"，然后在"设置"选项卡中"说明"。 </li><li>点击"查找组织"选项卡上。 </li><li>导入之前保存的参考图像。 </li><li>放置在舞台上的5微米厚的未染色FFPE幻灯片。移动台以相同的区域在参考图像中所描绘的。点击"插入图片"选项卡。 </li><li>单击"对齐"选项卡。使用鼠标和相关的箭头来参考图像调整到未染色的图像。 </li></ol> 4。幻灯片注释<ol><li>点击"注释"选项卡上。 </li><li>使用鼠标绘制一个围绕将显微切割的幻灯片所需区域的圆。 </li></ol> 5，载入耗材穆勒位<ol><li>填耗材轧机位具有所需缓冲液，此处PKD缓冲液中，通过拉动柱塞上下中的流体运动数次。请务必排除空气气泡。 </li><li>按"Z轴键"提高Z轴。 </li><li>加载耗磨位成机器。通过滑动耗材厂位到机器的顶部做到这一点，这样的白线在仪器上线与黑线耗材厂位。按"吸RESET"键，以便柱塞下降到正确的位置。注:这应该很容易滑动耗材厂位。如果不是，请确定缺口是否正确对齐。 </li><li>低Z轴再次按下"Z轴"按钮。 </li></ol> 6，解剖组织（ 图4） <ol><li>打开"解剖"选项卡。 </li><li>转电机和愿望速度为1。 </li><li>勾选"显示跟踪"复选框。注意:此允许用户在解剖过程中可视化的解剖区域。 </li><li>一旦准备解剖，继续举办"搞"按钮以及"抽吸＆＃8221;按钮的同时移动操纵杆以逆时针方向解剖组织。 </li><li>继续在反时针方向进行解剖，直到吸液是"满"标签变成红色。 </li></ol> 7，清空和删除耗材穆勒位<ol><li>放置一个0.5微升离心管耗材磨钻头的尖下。 </li><li>按"吸金"控制棒迅速弹出样品放入离心管中。 </li><li>新闻与增加力量弹出使用过的耗材厂位。 </li><li>通过拉动活塞回冲洗组织片段。推动活塞前进，以表达剩下的样品放入粉碎机易损位。所述组织片段现在将包含在微量离心管内。 </li></ol> 8，裂解样品用蛋白酶K消化热处理<ol><li>加入含5微克蛋白酶K至恢复组织样品70微升的TE pH值8.5。 </li><li>将样品放入可编程加热器振动筛。 </li><li>采用可编程加热器摇床在以下设定完全消化:60˚C，30分钟在1500转; 82˚C下15分钟在1500转; 25˚C为450转1分钟。样品现在即可下游应用。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis:+what+we+don't+know+can,+and+does,+hurt+us.">Tuberculosis: what we don't know can, and does, hurt us.</a> Science. 328 (5980), 852-856 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodissection+of+fibrous+tissue.">Mesodissection of fibrous tissue.</a> AvanSciBio. , (2013).</li><li>Paige, C., Bishai, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penitentiary+or+penthouse+condo:+the+tuberculous+granuloma+from+the+microbe's+point+of+view.">Penitentiary or penthouse condo: the tuberculous granuloma from the microbe's point of view.</a> Cell Microbiol. 12 (3), 301-309 (2010).</li><li>Mehra, S., Alvarez, X., Didier, P. 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G., Tyagi, S., Yoshimatsu, T., Scott, C., Ko, 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=Reduced+immunopathology+and+mortality+despite+tissue+persistence+in+a+Mycobacterium+tuberculosis+mutant+lacking+alternative+sigma+factor,+SigH.">Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH.</a> Proc Natl Acad Sci U S A. (12), 8330-8335 (2002).</li><li>Mehra, S., Kaushal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+reveals+extended+roles+of+the+Mycobacterium+tuberculosis+stress+response+factor+sigmaH.">Functional genomics reveals extended roles of the Mycobacterium tuberculosis stress response factor sigmaH.</a> J Bacteriol. 191 (12), 3965-3980 (2009).</li><li>Rohde, K. H., Veiga, D. F., Caldwell, S., Balazsi, G., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+the+transcriptional+profiles+and+the+physiological+states+of+Mycobacterium+tuberculosis+during+an+extended+intracellular+infection.">Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection.</a> PLoS Pathog. 8 (6), (2012).</li><li>Fontan, P. A., Voskuil, M. I., Gomez, M., Tan, D., Pardini, M., Manganelli, R., 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+Mycobacterium+tuberculosis+sigma+factor+sigmaB+is+required+for+full+response+to+cell+envelope+stress+and+hypoxia+in+vitro,+but+it+is+dispensable+for+in+vivo+growth.">The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.</a> J Bacteriol. 191 (18), 5628-5633 (2009).</li><li>Rustad, T. R., Harrell, M. I., Liao, R., Sherman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enduring+hypoxic+response+of+Mycobacterium+tuberculosis.">The enduring hypoxic response of Mycobacterium tuberculosis.</a> PLoS One. 3 (1), (2008).</li></ol>

作者透露，目前并无任何竞争经济利益。

Mesodissection是可以从所引起的病原体繁多病理病变的幻灯片被应用于RNA提取的技术。它是用户跟踪图像和正确对齐的图像是必要的。要做到这一点，该仪器必须跟随在成像软件的方向进行校正。解剖时，如果被解剖的兴趣幻灯片和面积不对齐，用户必须重新校准仪器。在剥离过程中的另一个重要步骤是加载自耗磨位的方式之前，夹层（步骤5.1），使得它是不含气泡。我们已经发现，气泡的存在降?...

The corresponding author was changed for the article, <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>. The corresponding author was changed from:
Teresa A. Hudock
to:
Deepak Kaushal

The corresponding author was changed for the article, <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>.

Erratum: A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

样品在传统上被手动从任一整个组织或使用针和解剖刀载玻片进行显微解剖。这就需要感兴趣的组织切片与周围组织部分2之间的明确分工。随着分子分析技术目前的进展，出现了越来越需要在细胞水平来评估组织。由于手工显微切割的局限性，已建立的技术包括液晶显示模块，以允许更大的隔离精度。这种技术允许研究者隔离从各种细胞和滑动类型的特定细胞群，然后其可用于下游分析，如基因表达分析。虽然非常有效的下游检测，液晶显示模块也不是没有限制的。首先，LCM是一个昂贵和费时的过程。此外，由于RNA的性质不稳定，它往往是具有挑战性的从LCM样品2获得高质量的RNA。由于diLCM的sadvantages，仍然需要在显微切割技术的新进展，使之更容易在一个合理的时间和敏感的方式更大一些研究人员。 在显微切割技术现在可一个这样的进步是被称为mesodissection的技术。在这种技术中一台机器是用来磨感兴趣的注解组织切片，并吸取它变成一种消耗品磨位3。下一步，该样品被吸入到一个集合管和用于下游应用。这个系统的优点是，它是显著成本较低和时间敏感。在我们的经验中，系统允许在十分钟内肺肉芽肿组织部分的剥离。在另一方面，传统的LCM将可能需要数小时才能完成该过程。该系统允许操作者装入一个参考幻灯片作为比较以及工具注解来使用。另外一个报告生成概述日E区被解剖的幻灯片。有两个主要的缺点mesodissection。虽然在提取多个单元格有效，它是具有挑战性的分离单个细胞。此外，所产生的图像的精度不明确的，因为如果使用其他成像显微镜。 结核病（TB）是人类的一个全球主要的传染病杀手，从感染的结果与结核杆菌 。在大多数暴露于结核分枝杆菌的气溶胶个体，这种感染潜伏的限制。在每年至少 ​​有10万人，它导致活动性肺结核病4。在潜伏感染， 结核杆菌包含称为肉芽肿病理性肺部病灶内。因此，它已被认为结核杆菌感染的结果决定于肉芽肿5的水平。 在这里，我们展示mesodissection如何可以用来microdissect引起结核杆菌肉芽肿。所使用的幻灯片是从被感染的恒河猴FFPE肺组织。此演示的目的，我们将剖析肉芽肿的全部。我们还证明，RNA可从回收的组织被提取。这种技术可以应用于组织样品从各种其他标本，然后用于各种下游检测的。

<table><tbody><tr><td>MeSectr</td><td>AvanSci Bio</td><td></td><td>Mesodissector</td></tr><tr><td>400 nm xScisors</td><td>AvanSci Bio</td><td></td><td>Other sizes available</td></tr><tr><td>THOR</td><td>AvanSci Bio</td><td></td><td>Programmable Heater-Shaker</td></tr><tr><td>NanoDrop2000</td><td>ThermoScientific</td><td>ND-2000</td><td></td></tr><tr><td>RNeasy FFPE extraction kit&amp;nbsp;</td><td>Qiagen</td><td>73504</td><td></td></tr><tr><td>Ovation RNA-Seq FFPE System&amp;nbsp;</td><td>Nugen</td><td>7150</td><td></td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td></td></tr></tbody></table>

a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas

显微切割已被用于组织的检查在DNA，RNA和蛋白水平超过十年。激光捕获显微术（LCM）是当今使用的最常见的显微切割技术。在该技术中，激光被用来灶性熔融的热塑性膜，其覆盖一个脱水的组织切片1。组织切片复合，然后抬起并从该膜分离。尽管这种技术可以成功地用于组织检查，这是耗时且昂贵的。此外，在成功完成使用这种技术的程序需要使用的激光的，从而限制了它的使用。所谓mesodissection一个新的更加实惠和实用显微切割方法是一种可行的解决方案，以LCM的陷阱。该技术采用了MESO-1/MeSectr系统轧机从幻灯片上的组织试样所需的组织，而同时分配和吸液至所需的组织样品恢复到耗材厂位。在解剖过程开始时，用户对齐福尔马林固定，石蜡包埋（FFPE）用苏木精伊红染色（H＆E）的参考滑盖滑动。此后，操作者诠释所需的解剖区域，并且前进到解剖相应的段。该程序生成的解剖的存档影像。 mesodissection的主要优点是解剖的滑动所需要的时间短，取平均10分钟从设置采样在该实验中产生。此外，该系统是显著更加成本有效的和用户友好。轻微缺点是，它并不像精确的激光捕获显微术。在这篇文章中我们将演示如何mesodissection可以使用从引起结核分枝杆菌（结核杆菌）FFPE肉芽肿幻灯片中提取的RNA。

上述协议显示了如何使用新的mesodissection技术从FFPE组织切片中提取的RNA。该协议的有效性是通过从NHP肺肉芽肿在各类感染阶段感染了结核杆菌的FFPE幻灯片所示。 图1-3是仪器。 图4的图像描绘了如何在解剖过程中发生，并通过该软件。 表1中生成的结果图像示出的RNA提取与来自NHP肉芽肿在每个感染状态的结果。 RNA扩增和纯化，以允许16秒（ 表2-4）<...

Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

显微切割已被广泛地用于DNA，RNA，和在组织内蛋白质的检查。激光捕获显微术（LCM）是最常用的方法，而是一种新的研磨技术，mesodissection，是最近可用。我们证明RNA提取从mesodissected福尔马林固定，石蜡的结核分枝杆菌肉芽肿包埋组织幻灯片。

一种新型显微切割方法恢复<em&gt;结核分枝杆菌</em&gt;从福尔马林固定，石蜡包埋肺肉芽肿特异性转录

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

a-novel-microdissection-approach-to-recovering-mycobacterium

Research

JoVE Journal

Immunology and Infection

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

8.0K 次观看.  Tulane National Primate Research Center. 显微切割已被广泛地用于DNA，RNA，和在组织内蛋白质的检查。激光捕获显微术（LCM）是最常用的方法，而是一种新的研磨技术，mesodissection，是最近可用。我们证明RNA提取从mesodissected福尔马林固定，石蜡的结核分枝杆菌肉芽肿包埋组织幻灯片。 

视频: A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

科研

遗传学

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

癌症研究

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

免疫与感染

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

结核病分子细菌负荷测定（TB-MBLA）

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

医学

<meta charset="utf8"></head><body>保留肺部真菌孢子定位以进行荧光显微镜分析

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

<meta charset="utf8"></head><body>保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

生物学

使用琼脂糖包埋的振动切片机进行创新的肺部成像

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

作者聚焦:使用琼脂糖包埋的肺组织切片研究空间蛋白表达 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

一种新型显微切割方法恢复

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此视频中的章节

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

结核病分子细菌负荷测定（TB-MBLA）

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片