Name: the use of ex vivo whole-organ imaging and quantitative tissue histology to determine the bio-distribution of fluorescently labeled molecules
Uploaded: 2016-12-24T15:00:00
Description: Watch this Scientific Journal Video about The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules at JoVE.com

Partial salary support for GLB is provided by NIH grant R01HL121527. JWDM is supported by the Currier Fellowship in Neurology.

注：在本协议中的所有动物的使用是经密西西比医学中心大学的机构动物护理和使用委员会。 1.动物和组织处理<ol><li>管理和牺牲<ol><li>麻醉，用3％异氟烷将动物5分钟，并通过观察不存在脚趾捏反射的验证麻醉深度。应用兽医软膏眼睛防止干燥时的麻醉下。 </li><li>称重动物并吸取或罗丹明标记的ELP，SYNB1-ELP，或达-ELP的50毫克/公斤的剂量为1.5毫升管中。减100微升施用标记的?...

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Biol. 7 (5), 626-634 (2003).</li><li>Vugmeyster, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+toxicology+of+therapeutic+proteins:+Advances+and+challenges.">Pharmacokinetics and toxicology of therapeutic proteins: Advances and challenges.</a> World J. Biol. Chem. 3 (4), 73 (2012).</li><li>Stumpf, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+and+microscopic+autoradiography+to+determine+tissue+distribution+of+biopharmaceuticals+-+Target+discoveries+with+receptor+micro-autoradiography+engendered+new+concepts+and+therapies+for+vitamin+D.">Whole-body and microscopic autoradiography to determine tissue distribution of biopharmaceuticals - Target discoveries with receptor micro-autoradiography engendered new concepts and therapies for vitamin D.</a> Adv. Drug Deliv. 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而离体全器官成像一般是直接的，粘附到一些基本概念和技术可以改善生物分布测量的准确度。的光的经验短的波长在大多数组织中高度的散射和吸收的，这显著影响短波长的荧光团的效用。这些荧光限制了深部组织研究中的应用，但他们已在实验中有效地用于寻找表面组织或进入眼内。相反，光的长波长经受显著较少的散射和吸收，造成更大的渗透和具有较厚组织10更准确的结果...

荧光标记是用于确定化合物的生物分布，使用一系列的设备是共同的许多实验室一个容易使用的，有效的方法。荧光团是广泛可用的，相对便宜的，以及各种波长，使得多个标记分子可以同时使用无干扰的进入。大多数荧光团具有一系列化学缀合到目标化合物不同的活性基团的，和缀合的过程通常是简单的为大多数类型的反应性位点的。此外，对于荧光标记的化合物的测量所需的设备在许多实验室是常见的。荧光显微镜，成像仪，和滑动扫描器都可以在不同的情况下使用，使得使用荧光标记的高度可访问的。荧光标记经常用于确定在体内和离VIV化合物的生物分布和动力学Ø与实时成像设备，如IVIS光谱成像仪，并通过定量组织组织学2,3。 离体全器官成像的使用实时成像设备的使用增加了随着时间的推移，由于其易于使用和快速创建标记的化合物的相对浓度的准确的比较，而无需tissues4的进一步处理能力。然而，虽然离体全器官成像可以允许容易分析和比较，它不产生一个组织内绝对化合物浓度的定量测量。这是由于完整的器官内的光散射的影响。由于光散射（以及在较小程度上，吸光度）由组织的大小和密度而变化，整个器官成像能够低估在大型或密集的器官组织水平。制定相应的标准绝对浓度测量也很困难，因为我们必须模仿厚度和每个器官的密度。另一方面，整个器官成像获得的试剂的相对组织水平的快速方法，它是理想的比较多个相关分子的相对生物分布（例如，在药物靶向研究）。一种替代的策略是利用荧光定量组织学，从定量放射自显影的方法中派生的技术，以获得测试试剂5,6-绝对组织水平。而不是将整个动物或器官成为一个想象设备，定量组织组织学要求每个组织切片，固定在载玻片上，扫描，单独分析。测试剂的标准制备并在相同的厚度器官样品切片。通过切割所有器官和标准，以相同的厚度，由于光散射或吸收可变性被消除，和组织的荧光强度可以配合到标准曲线来确定绝对concent配给。虽然这种方法中，当正确完成，是定量的，它也是劳动密集的，容易处理不当。给出定量组织学和劳动的显著成本较高的更复杂的性质相比整个器官成像时，它成为值得研究，其中每个过程是最实际的检验荧光标记的化合物的生物分布时使用。这个协议提供了如何这些方法可以一起使用，以有效地比较罗丹明标记的弹性蛋白样多肽（ELP）的生物分布，有或没有加入SYNB1或达细胞穿透肽的详细描述，通过鼻内（IN）和静脉内（IV）给药途径。

<table border="0" cellpadding="0" cellspacing="0" width="819">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Reagents</td>
			<td style="width:99px;"></td>
			<td style="width:159px;"></td>
			<td style="width:332px;"></td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:231px;">Maleimide derivitized fluorophors (e.g. tetramethylrhodamine-5-maleimide, AlexaFluor 633-C5-maleimide)</td>
			<td style="width:99px;">Thermo Fisher</td>
			<td style="width:159px;">T6027, A20342</td>
			<td>Thiol reactive fluorescent dyes for protein labeling</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Phosphate Buffered Saline</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:159px;">1002243569</td>
			<td>PBS Buffer for rinsing</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:231px;">Optimal Cutting Temperature Compound</td>
			<td style="width:99px;">Tissue-Tek</td>
			<td style="width:159px;">4585</td>
			<td>Used for freezing and mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Equipment</td>
			<td style="width:99px;"></td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">IVIS Spectrum</td>
			<td style="width:99px;">Perkin Elmer</td>
			<td style="width:159px;"></td>
			<td>For ex vivo whole organ imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cryomicrotome</td>
			<td style="width:99px;">Thermo</td>
			<td style="width:159px;"></td>
			<td>For cryosectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fluorescence slide scanner</td>
			<td style="width:99px;">Perkin Elmer</td>
			<td style="width:159px;"></td>
			<td>For slide scanning</td>
		</tr>
	</tbody>
</table>

the use of ex vivo whole-organ imaging and quantitative tissue histology to determine the bio-distribution of fluorescently labeled molecules

荧光标记是用于在各种体外和体内实验条件下检验标记的分子的命运成熟过程。荧光探针在确定施用大分子的生物分布，其中，除了一小分子荧光标记的是不太可能影响的动力学或化合物的生物分布是特别有用的。多种方法可用来检验生物分布，在努力所需的显著变化，并且所得到的测量结果是否完全定量的，但是使用在结合多个方法可以提供用于分析生物分布的快速和有效的系统。 离体全器官成像是可以用于对组织内和多种类型的组织或治疗组之间快速比较荧光分子的相对浓度的方法。使用成像开发平台无需进一步处理决定的形式完整的组织内专为活的动物或整个器官成像，荧光，省时省力，同时提供全面的生物分布的准确描述。这个过程是在实验试图确定的化合物的组织特异性或多个不同的化合物的比较理想的。另一方面定量组织组织学要求，以创建标记的化合物的定量测量组织的广泛进一步处理。准确评估生物分布，所有感兴趣的组织必须被切片，扫描，以使组织或组之间的比较相对于标准曲线进行分析。定量组织病理是确定组织中的绝对化合物浓度的黄金标准。 在这里，我们描述了如何两种方法可以一起有效地用于评估不同的给药方法的能力第二化合物修饰靶向和递送荧光标记的分子在中枢神经系统1。

下面的数据说明三种化合物的递送：被称为ELP和ELP的两个版本与细胞穿透肽（SYNB1-ELP和康达-ELP）9修饰的药物递送载体。所有这三个化合物标记四甲-5-马来酰亚胺和通过两个给药途径交付（IN和IV）。这些实验的目的是确定将导致最好渗透到中枢神经系统（CNS）3，其化合物和给药途径。 图1A显示代表离体从处理过的动物的器官的整个器官的?...

Watch this Scientific Journal Video about The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules at JoVE.com

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

离体全器官成像是用于确定内和组织或治疗组之间的荧光标记的化合物的相对浓度快速方法。相反，定量荧光组织学，而更多的劳动力密集的，允许对标记分子的绝对组织水平的量化。

指某东西的用途<em&gt;离体</em&gt;全器官成像和定量组织组织学确定荧光标记分子的生物分布

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Research

JoVE Journal

Biology

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

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 ...

科研

生物学

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...