Name: spot the correct tissue every time in multi-tissue blocks
Uploaded: 2015-05-31T14:00:00
Description: Watch this Scientific Journal Video about SpOT the Correct Tissue Every Time in Multi-tissue Blocks at JoVE.com

作者要感谢布伦特·哈里斯博士提供稿件的严格审查。这些研究在隆巴迪综合癌症中心组织病理与组织共享资源是支持由NIH / NCI资助P30-CA051008部分进行。内容完全是作者的责任，并不一定代表美国国家癌症研究所和美国国立卫生研究院的官方意见。

1.施工现场的<ol><li>添加50毫克牛血清白蛋白（BSA）的1毫升组织标记染料在15ml锥形管或2 ml离心管，涡旋1分钟，或直至完全溶解。 注意：对于这个协议，使用生化级BSA。而其他等级和纯度都没有经过测试，预计品位和纯度不会对现货的成功显著的影响。 </li><li>热9毫升羟乙基在15ml的锥形管在微波中，在30％的功率，持续10秒的增量琼脂糖处理凝胶直到凝胶完全融化。 </li><li>结合熔融加工的凝...

<ol>
	<li>Jawhar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarray:+a+rapidly+evolving+diagnostic+and+research+tool.">Tissue microarray: a rapidly evolving diagnostic and research tool.</a> Ann. Saudi. Med. 29 (2), 123-127 (2009).</li><li>Dhir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays:+an+overview.">Tissue microarrays: an overview.</a> Methods Mol. Bio. 441, 91-103 (2008).</li><li>Parsons, M., Grabsch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+tissue+microarrays.">How to make tissue microarrays.</a> Diagn. Histopath. 15 (3), 142-150 (2009).</li><li>Saxena, R., Bade, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+7+Tissue+Microarray+–+Construction+and+Quality+Assurance.">Ch. 7 Tissue Microarray – Construction and Quality Assurance.</a> Immunohistochemical Staining Methods 6th ed. , (2013).</li><li>Nocito, A., Kononen, J., Kallioniemi, O., Sauter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays+(TMAs)+for+high-throughput+molecular+pathology+research.">Tissue microarrays (TMAs) for high-throughput molecular pathology research.</a> Int. J. Cancer. 94, 1-5 (2001).</li><li>Rangel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tissue+microarray:+helpful+hints.">The tissue microarray: helpful hints.</a> J.Histotech. 25 (2), 93-100 (2002).</li><li>Hammer, A., Williams, B., Dietz, H., Hamilton-Dutoit, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+immunophenotyping+of+43+ferret+lymphomas+using+tissue+microarray+technology.">High-throughput immunophenotyping of 43 ferret lymphomas using tissue microarray technology.</a> Vet. Path. 44, 196-203 (2007).</li><li>Eckel-Passow, J., Lohse, C., Sheinin, Y., Crispen, P., Krco, C., Kwon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays:+one+size+does+not+fit+all.">Tissue microarrays: one size does not fit all.</a> Diagn Pathol. 5, 48-57 (2010).</li><li>Lin, Y., Hatem, J., Wang, J., Quinn, A., Hicks, D., Tang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarray-based+immunohistochemical+study+can+significantly+underestimate+the+expression+of+HER2+and+progesterone+receptor+in+ductal+carcinoma+in+situ+of+the+breast.">Tissue microarray-based immunohistochemical study can significantly underestimate the expression of HER2 and progesterone receptor in ductal carcinoma in situ of the breast.</a> Biotech. Histochem. 86 (5), 345-350 (2011).</li><li>Wampfler, 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=Determining+the+optimal+numbers+of+cores+based+on+tissue+microarray+antibody+assessment+in+non-small+cell+lung+cancer.">Determining the optimal numbers of cores based on tissue microarray antibody assessment in non-small cell lung cancer.</a> J Cancer Sci. Ther. 3, 120-124 (2011).</li><li>Quagliata, L., Schlageter, M., Quintavalle, C., Tornillo, L., Terracciano, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+new+players+in+hepatocarcinogenesis:+Limits+and+opportunities+of+using+tissue+microarray+(TMA).">Identification of new players in hepatocarcinogenesis: Limits and opportunities of using tissue microarray (TMA).</a> Microarray. 3, 91-102 (2014).</li><li>Miettinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+generating+multitissue+blocks+without+special+equipment.">A simple method for generating multitissue blocks without special equipment.</a> Immunohistochem. Mol. Morphol. 20 (4), 410-412 (2012).</li><li>Jones, M., Calabresdi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar-gelatin+for+embedding+tissues+prior+to+paraffin+processing.">Agar-gelatin for embedding tissues prior to paraffin processing.</a> BioTechniques. 42, 569-570 (2007).</li><li>Carson, F., Hladik, 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> Histotechnology: A Self-Instructional Text. , (2009).</li><li>Suvarna, S., Layton, C., Bancroft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bancroft&#8217;s Theory and Practice of Histological Techniques. 7th ed. , (2013).</li></ol>

成功制备的斑点和利用需要认真遵守一些技术步骤。羟乙基琼脂糖熔化应缓慢并且在低热完成。在高的热迅速熔化可导致琼脂糖一些击穿不到最佳效果。当切割染料载货插头插入件进行处理，确保每一块的厚度大于4.5毫米，不超过7 mm。的圆形端，因为它们不是均匀5mm的厚度被除去废物。件小于5mm将导致该就有可能被用尽之前由邻近组织材料用尽切片的创建短斑点。为了确保现场出现过块的整体，...

嵌入在一个单一的石蜡块从多个个体的组织样品的能力使得护理和个人之间的容易的侧方比较，消除了幻灯片之间的变异，并降低切片和染色标本的成本和工作量。这些多组织块通常产生含有在非阵列布局从多个个体组织或者组织微阵列（TMA）或石蜡块。样本标识的保养是任何多组织分析的成功是至关重要的。使用的TMA因为它们的发展，提高分析的效率，减少幻灯片之间变化，节省宝贵的组织资源，并减少的时间和实验1成本研究者已经。的TMA的正确取向可以采用多种方法来完成，包括组织核心2-4，芯组的非对称布置3，5（ 例如，CONTRO之间空格，行或列升与处理），“信标”芯6，以及位于该TMA基质3外指定取向芯。虽然这些方法工作以及组织芯片，​​最消耗宝贵的核心TMA空间和组织芯片与差距和空间作为标识符可能会造成混乱的组织核心耗尽了时间。此外，这些方法不适合于在多组织块使用未经阵列形式，因为它们依赖于均匀微阵列芯作为标识符的紧密有序图案异常。大多数非排列多组织块必须适应非均匀组织，根据定义，不显示所述结构化阵列网格使这些象差立场标。 虽然有许多优点，使用一个TMA，最大缺点之一是芯的小尺寸，这并不总是代表很大程度上异质组织1。非排列的多块组织提供了许多的t他好处TMA的，但包含较大的组织样本，或从动物研究整个器官。使用单核组织微阵列具有不同的一致性与基于感兴趣蛋白质整个组织切片和许多需要多个内核以提高一致性7-11。由于复杂性和一些生物标志物的表型异质性，的TMA使用甚至大量芯（&gt; 10）仍然是不充分，并且可能需要的方法比微阵列分析8等。此外，TMA建设费时，技术要求高，需要初始投资成本与投资或访问一个组织阵列。非阵列多的组织块可在任何基本的实验室有显著更少的时间和精力，是需要更多的组织比阵列允许或作为一种手段来削减成本和简化的分析研究，一个有效的替代。 非排列的多块组织同样需要一个可靠的或ientation标记来跟踪样品标识，然而，这些标志物的开发已受到了限制。多描述组织取向的文献集中在正确生理取向为嵌入个人组织片，如纹身的组织与墨12，在处理前13预嵌入琼脂明胶组织，和标记的某些组织与槽口14或缝线15 。虽然功能性的，这些方法是不理想的因为在多组织块标记由于其局限性。缝线将通过快速切片，可能不会在每个部分可见。预嵌入用琼脂明胶可以保持在适当的取向的组织处理和嵌入期间技术，但不提供视觉提示在石蜡块和幻灯片多个样品之间进行区分。在组织上凹口或染料可以复杂化分析或阻塞重要形态细节。另外，组织认同在非排列多组织块可以通过组织片段中的非对称布置嵌入保持，但是这需要3个或更多的组织碎片和可能不允许组织用于分析的最佳安排。 试样方向标记（点）已发展为一种简单，廉价的方法清楚地标识在多组织块组织，并提供比现有方法的方向很多好处。现场是一个小的，丰富多彩的，核心组成羟乙基琼脂糖凝胶处理和组织标记染料，并渗透石蜡（ 图2C）。现货内核嵌入在结束旁边的一个单一的组织在多组织石蜡块，并显示为块，并在每一个部分鲜艳的点（ 图1A - D），清楚地表明了块部分的正确方向为便于组织鉴定。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HistogelTM Specimen Processing Gel</td>
			<td>Thermo Scientific</td>
			<td>HG-4000-012</td>
			<td><a href="http://www.thermoscientific.com/en/product/richard-allan-scientific-histogel-specimen-processing-gel.html">http://www.thermoscientific.com/en/product/richard-allan-scientific-histogel-specimen-processing-gel.html&#160;</a></td>
		</tr>
		<tr>
			<td>Tissue Marking Dye</td>
			<td>Triangle Biomedical Sciences, Inc.</td>
			<td>TMD-5</td>
			<td>Any tissue marking dye would most likely be sufficient.</td>
		</tr>
		<tr>
			<td>Arraymold Kit A 2 mm (60 core)</td>
			<td>Arraymold</td>
			<td>20015A</td>
			<td>Any manual tissue arrayer would work similarly.</td>
		</tr>
	</tbody>
</table>

spot the correct tissue every time in multi-tissue blocks

多组织石蜡块提供高通量分析以更高的效率，实验的均匀性，并且减少了时间和成本。组织芯片弥补大多数多组织石蜡块，但越来越多的研究人员正在使用含多个人，可以提供很多的组织芯片的优点，但没有规划和设备的大量投资较大的组织非摆着块。任何多组织分析的关键部件是用于识别每个单独的组织的取向方法。虽然方法存在以维持适当的取向，并识别在多组织块的组织，大多不是非常适合于非排列的块，可在阵列内消耗宝贵的空间和/或难以生产在标准组织学实验室。试件方向标签（SPOT）是一种简单，低成本的定向工具，这显然是在石蜡块和所有的组织切片可见FO- [R可靠的标本鉴定阵列和非阵列布局。当场提供了超过现有的取向方法用于非排列块的优点，因为它不需要任何直接修改到组织，并允许在组织片的布置的灵活性。

当场显示为圆形，在石蜡块色彩鲜艳点（ 图1A和图2D），在所有的石蜡切片，并通过H＆E或IHC染色程序保留在玻璃载片上（ 图1B - 1D和2D）。这种明显的视觉提示助剂既在识别每个单独的组织片的组织学技术人员和研究人员，并简化通信作为组织学技术人员可以使用该视觉提示来指示组织片的研究者从幻灯片在显微镜收集数据的排列。?...

Watch this Scientific Journal Video about SpOT the Correct Tissue Every Time in Multi-tissue Blocks at JoVE.com

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

试件定位标记（点）的目的是充当定向工具在单个组织识别在多组织石蜡块中提供帮助。这些协议表明它是如何容易地从普通的，低成本的组织学材料构成，并作为在石蜡块和切片的可靠视觉标记。

现货正确的组织多组织块每一次

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...

Research

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Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

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Implantation of Engineered Tissue in the Rat Heart

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Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

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In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

在出生后的小鼠听泡解剖：中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

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Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

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