Name: sivq-lcm protocol for the arcturusxt instrument
Uploaded: 2014-07-23T14:00:00
Description: Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

这项研究是由美国国家癌症研究所癌症研究中心院内研究计划美国国立卫生研究院，部分支持。

所描述的协议是采用按照关于使用人体组织样本的美国国立卫生研究院的规则。 1，组织准备<ol><li>前年初，根据机构审查委员会（IRB）的协议得到人体组织标本。 </li><li>选择组织/细胞块的类型和相应的处理方法[福尔马林固定石蜡包埋（FFPE），冷冻，或乙醇固定，石蜡包埋（EFPE）]。福尔马林固定提供了最佳的组​​织学，随后用乙醇固定，并快速冷冻。然而，固定和组?...

<ol>
	<li>Bonner, R. 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Emmert-Buck, M. 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=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Edwards, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+mammalian+tissue.">Laser capture microdissection of mammalian tissue.</a> J Vis Exp. (8), (2007).</li><li>El-Serag, H. B., 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=Gene+expression+in+Barrett's+esophagus:+laser+capture+versus+whole+tissue.">Gene expression in Barrett's esophagus: laser capture versus whole tissue.</a> Scandinavian journal of gastroenterology. 44, 787-795 (2009).</li><li>Espina, V., 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature. 1, 586-603 (2006).</li><li>Harrell, J. C., Dye, W. W., Harvell, D. M., Sartorius, C. A., Horwitz, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contaminating+cells+alter+gene+signatures+in+whole+organ+versus+laser+capture+microdissected+tumors:+a+comparison+of+experimental+breast+cancers+and+their+lymph+node+metastases.">Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases.</a> Clinical &amp; experimental metastasis. 25, 81-88 (2008).</li><li>Rodriguez-Canales, 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=Optimal+molecular+profiling+of+tissue+and+tissue+components:+defining+the+best+processing+and+microdissection+methods+for+biomedical+applications.">Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.</a> Methods in molecular biology. 980, 61-120 (2013).</li><li>Silvestri, 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=Protein+pathway+biomarker+analysis+of+human+cancer+reveals+requirement+for+upfront+cellular-enrichment+processing.">Protein pathway biomarker analysis of human cancer reveals requirement for upfront cellular-enrichment processing.</a> Laboratory investigation; a journal of technical methods and pathology. 90, 787-796 (2010).</li><li>Eberle, F. 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=Immunoguided+laser+assisted+microdissection+techniques+for+DNA+methylation+analysis+of+archival+tissue+specimens.">Immunoguided laser assisted microdissection techniques for DNA methylation analysis of archival tissue specimens.</a> The Journal of molecular diagnostics : JMD. 12, 394-401 (2010).</li><li>Kim, H. 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=Distinctions+in+gastric+cancer+gene+expression+signatures+derived+from+laser+capture+microdissection+versus+histologic+macrodissection.">Distinctions in gastric cancer gene expression signatures derived from laser capture microdissection versus histologic macrodissection.</a> BMC medical genomics. 4, 48 (2011).</li><li>Klee, E. W., 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=Impact+of+sample+acquisition+and+linear+amplification+on+gene+expression+profiling+of+lung+adenocarcinoma:+laser+capture+micro-dissection+cell-sampling+versus+bulk+tissue-sampling.">Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling.</a> BMC medical genomics. 2, 13 (2009).</li><li>Zheng, J., Garg, S., Wang, J., Loose, D. S., Hauer-Jensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissected+mucosa+versus+whole+tissue+specimens+for+assessment+of+radiation-induced+dynamic+molecular+and+pathway+changes+in+the+small+intestine.">Laser capture microdissected mucosa versus whole tissue specimens for assessment of radiation-induced dynamic molecular and pathway changes in the small intestine.</a> PloS one. 8, e53711 (2013).</li><li>Boone, D. R., Sell, S. L., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+enriched+populations+of+neurons+or+single+neurons+for+gene+expression+analysis+after+traumatic+brain+injury.">Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.</a> J Vis Exp. (74), (2013).</li><li>Iyer, E. P., Cox, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+Drosophila+peripheral+neurons.">Laser capture microdissection of Drosophila peripheral neurons.</a> J Vis Exp. (39), (2010).</li><li>Hipp, 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=SIVQ-aided+laser+capture+microdissection:+A+tool+for+high-throughput+expression+profiling.">SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling.</a> Journal of pathology informatics. 2, 19 (2011).</li><li>Hipp, J. D., Cheng, J. Y., Toner, M., Tompkins, R. G., Balis, U. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+Invariant+Vector+Quantization:+A+pattern+matching+algorithm+for+multiple+classes+of+image+subject+matter+including+pathology.">Spatially Invariant Vector Quantization: A pattern matching algorithm for multiple classes of image subject matter including pathology.</a> J Pathol Inform. 2, 13 (2011).</li><li>Hipp, 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=Optimization+of+complex+cancer+morphology+detection+using+the+SIVQ+pattern+recognition+algorithm.">Optimization of complex cancer morphology detection using the SIVQ pattern recognition algorithm.</a> Anal Cell Pathol (Amst). , (2011).</li><li>Hipp, 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=Integration+of+architectural+and+cytologic+drive+n+image+algorithms+for+prostate+adenocarcinoma+identification.">Integration of architectural and cytologic drive n image algorithms for prostate adenocarcinoma identification.</a> Analytical cellular pathology. 35, 251-265 (2012).</li><li>Hipp, 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=Automated+area+calculation+of+histopathologic+features+using+SIVQ.">Automated area calculation of histopathologic features using SIVQ.</a> Anal Cell Pathol (Amst. 34, (2011).</li><li>Cheng, 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=Automated+vector+selection+of+SIVQ+and+parallel+computing+integration+MATLAB:+Innovations+supporting+large-scale+and+high-throughput+image+analysis+studies.">Automated vector selection of SIVQ and parallel computing integration MATLAB: Innovations supporting large-scale and high-throughput image analysis studies.</a> Journal of pathology. 2, 37 (2011).</li><li>Roy Chowdhuri, 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=Semiautomated+laser+capture+microdissection+of+lung+adenocarcinoma+cytology+samples.">Semiautomated laser capture microdissection of lung adenocarcinoma cytology samples.</a> Acta Cytol. 56, 622-631 (2012).</li><li>Hipp, 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=Image+Microarrays+(IMA):+Digital+Pathology's+Missing+Tool.">Image Microarrays (IMA): Digital Pathology's Missing Tool.</a> Journal of pathology. 2, (2011).</li><li>Hanson, J. 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=Expression+microdissection+adapted+to+commercial+laser+dissection+instruments.">Expression microdissection adapted to commercial laser dissection instruments.</a> Nature. 6, 457-467 (2011).</li></ol>

我们提出了一个协议，用于SIVQ-LCM的应用，以microdissect从FFPE人体乳腺组织免疫染色上皮细胞。使用图像分析算法，如SIVQ，降低了实际操作所需的显微切割过程的时间的量。这对于该领域的潜在的重要进步，因为操作者的时间和精力，通常为所关注的细胞的精确解剖中的限速步骤。在本协议中，我们特别适合我们的程序到ArcturusXT仪器，虽然它很可能将有可能适应SIVQ等算法其他市售的显微仪器，以?...

最初开发于90年代中期，激光捕获显微切割（LCM）使用户通过微观可视化1，2，精确捕捉从组织学组织切片特定的细胞或细胞区域。比较LCM的分子分析与组织擦伤许多研究说明了该方法3-12的值。此外，也有关于可用于观看13,14的技术3视频协议的出版物。然而，尽管它被证明价值，LCM可以是繁琐和费力的，当感兴趣的目标是一个分散的细胞群体中的异质组织切片时，或者当需要进行特定的下游应用，如蛋白质组学大量的细胞。放置在操作人员的负担，导致我们结合一个强大的图像分析算法，以指导对LCM进程15制定LCM半自动夹层的方法。 &lt;P类=“j​​ove_content”&gt;在与密歇根大学合作，我们的实验室在NIH扩展先前制定和报告的空间不变的矢量量化（SIVQ）算法的方式，使其到半自动化的组织选择过程中固有的引导显微切割，从而使可用在头脑里的病理学家或生命科学家的工具。空间不变的矢量量化（SIVQ）是一种算法，允许用户简单地“点击”感兴趣的组织学特征来创建一个环载体（谓词图像特征），可以用来搜索整个组织学图像，调整阈值的统计根据需要16-21。由此产生的热图显示匹配的质量初始谓词图像特征，并随后被转换成一个单一的颜色，可以导入到LCM仪器（红色）标注地图。自动选型软件，AutoScanXT，然后用于绘制地图的基础上SIVQ的注解引导从组织样品中的靶细胞的捕获。下面详细的协议描述的实施SIVQ进入显微切割工作流程。

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Positive Charged Glass Slides</td>
			<td style="width:84px;">Thermo Scientific</td>
			<td style="width:119px;">4951Plus-001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Xylenes, ACS reagent, &#8805;98.5% xylenes + ethylbenzene basis&#160;</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">247642</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous</td>
			<td style="width:84px;">The Warner-Graham Company</td>
			<td style="width:119px;">6.505E+12</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Arcturus CapSure Macro LCM Caps</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">LCM0211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">ArcturusXT Laser Microdissection Instrument</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">ARCTURUSXT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">AutoScanXT Software</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:216px;">Spatially Invariant Vector Quantization (SIVQ)</td>
			<td style="width:84px;">University of Michigan</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]</td>
		</tr>
	</tbody>
</table>

sivq-lcm protocol for the arcturusxt instrument

SIVQ-LCM是一种新的方法，它可以自动和简化了较为传统的，依赖于用户的激光剥离过程。它的目的是建立一个先进的，快速定制的激光剥离技术平台。在这份报告中，我们描述了图像分析软件空间不变的矢量量化（SIVQ）整合到ArcturusXT仪器。该ArcturusXT系统包含一个红外（IR）和紫外（UV）激光，允许特定的细胞或大面积解剖。主要目标是提高速度，精度，激光剥离，以提高样品通量的重现性。这种新方法有利于动物和在研究和临床工作流程人体组织的显微切割。

一个FFPE人体乳腺组织切片的免疫染色用标准IHC协议23细胞角蛋白AE1/AE3。染色后，将组织切片放置在ArcturusXT阶段，如上述的SIVQ-LCM协议发起。自组织不能盖玻片为显微切割的IHC +染色的细胞可以是难以辨别视觉（ 图1A）。因此，为了提供更好的折射率匹配和改进的图像，二甲苯加入到该组织部分来创建一个伪盖玻片15（ 图1B）。 JPEG图像，然后拍摄的伪盖片?...

Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

SIVQ-LCM Protocol for the ArcturusXT Instrument

为ArcturusXT仪器SIVQ-LCM协议

SIVQ-LCM是一种创新的方法，运用计算机算法，空间不变的矢量量化（SIVQ），驱动激光捕获显微切割（LCM）的过程。该SIVQ-LCM流程大大提高显微切割的速度和精度，以在这两个研究和临床应用中的设置。

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

Research

JoVE Journal

Biology

Protocol for Mosquito Rearing (A. gambiae)

Protocol for Mosquito Rearing A. gambiae

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

科研

生物学

Neutrophil Isolation Protocol

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Protocol for the Production of KLRG1 Tetramer

KLRG1四聚体的生产协议

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

用于图像分析的时钟扫描协议：ImageJ插件

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

一种省力和可重复的触摸力信号突变屏幕协议,用于研究阿拉伯植物示范植物的三角性发生

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

用于映射植物根系架构特征的简单协议

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...