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>结合熔融加工的凝胶和染料BSA溶液在单一的15毫升锥形管中，并用移液管充分混合。涡的解决方案。 </li><li>放置锥形管在冰箱中几个小时或直至固体。 </li><li>使用长而细，金属铲或探针从锥形管轻轻松开彩色凝胶塞。 </li><li>使用手术刀或刀片来CUt是凝胶横插分成5毫米厚的切片。取出圆端和处置废物（ 图2A）。 </li><li>将凝胶塞部分平面中组织学盒并保持在70％乙醇中1小时。至少3的变化，在24小时内发生变化的70％的乙醇溶液每2-4小时。 </li><li>手动处理通过乙醇系列（1小时每凝胶部分:70％乙醇，80％乙醇，95％乙醇，100％乙醇（3×的变化），然后清除在清除溶液3次1小时的每个（ 例如，脂族烃（二甲苯替代））在这个协议中使用，二甲苯也可以接受），并渗透有熔融石蜡（3×为每1小时），可以手动或以组织processer。 注:在充分脱水在100％乙醇中，染料可能会泄漏从凝胶塞这可能会影响在自动组织处理器其它组织和液体试剂。因此，人工补液强烈建议，但自动化处理EQU盟友有效。 </li><li>嵌入多个处理凝胶切片用标准石蜡包埋方法（ 图2B）。允许块冷却并硬化，然后回复到室温。 </li><li>使用所需尺寸的真皮冲针从嵌入凝胶切片，直至该块被耗尽（ 图2C）除去斑核。单个块可提供高达20×2平方毫米的核心。存储内核在阴凉处。使用真皮冲头在这个协议中看到图中，在1.5毫米（ 图1D和2D）或2毫米（ 图1B，1C和2C）。 </li></ol> 2.使用斑点东方非阵列块<ol><li>创建组织取向图以指示所希望的位置和所有单独的组织块要被嵌入在一起的身份，和光点的位置。 注:该组织的方向图是一本图文并茂的地图，包括p所有的组织块标有标识符和点的位置的位置物质环境。它可以以电子方式创建，也可以手工绘制。该图应使用任何方法最适合使用的最终产品的技术人员和研究人员来创建。此地图将作为研究者导向，以便样品可以在微观分析容易地识别。在手绘本协议中使用的地图。它描绘了与每片组织和绘制并标记在相同的位置的实际组织块和滑动当场组织块和显微镜载玻片。 </li><li>处理组织片通常，以确保它们保持正确整个票房和处理步骤识别。此协议，处理该组织1小时每中:70％的乙醇，80％乙醇，95％乙醇，100％乙醇（×3的变化），脂族烃（二甲苯代替）（×3的变化），和熔融的组织浸润石蜡在60°C（X3的变化）。 注意:LL组织应按照标准组织学实验室程序进行处理。这可以是可变的，由于各个实验室过程，组织大小和类型，但是这将不会影响到使用点作为定向工具的能力。 </li><li>根据该组织的取向图安排组织片的嵌入站上以正确的分配位置。使用方向组织图作为参考来指导如何安排，当场组织块每块技术员。 注意:在这个协议中嵌入技师把手绘取向图旁边嵌入台和布置在组织片的嵌入站上完全相同的结构所绘制在图上。这确保了所有组织片留在最终产物中正确地识别。这是成功使用多组织块的绝对关键。 </li><li>确保当场核心是身高超过所有组织块被嵌入在该块。 </li><li>根据使用标准嵌入方法组织方向图嵌入的组织块，然后在年底（横向）的地点在合适的位置。根据需要，召开现场会（S）直立钳，直到石蜡凝固，以至于现场（S）也不会摔倒（1-2分钟）。 </li><li>科和染色石蜡切片使用标准方法的地方。 <ol><li>允许部分浮在水浴中于40℃和粘附到带正电荷的载玻片（不带电荷的载玻片尚未经过测试）。干燥的干燥炉的幻灯片，在60℃下进行至少20分钟。 </li><li>将载玻片在自动染色机和加工通过下列试剂:二甲苯（二甲苯1-5分钟，二甲苯2和3-2分钟，每），100％乙醇（2分钟），95％乙醇（1分钟），80 ％乙醇（30秒），70％的乙醇（30秒），自来水（2分钟），苏木（1.5分），自来水（3分钟），酸醇（1分钟），自来水（2分钟），BLuing试剂（1分钟），自来水（2分钟），80％的乙醇（30秒），曙红（1分钟），自来水（10秒），80％乙醇（1分钟），100％乙醇（3变化，每2分钟），二甲苯（3变化，每30秒）。 </li><li>盖上玻璃盖玻片，安装有安装介质的幻灯片。 注:在这个协议中，组织学技术人员在切片5微米的部分上切片的石蜡块。对于这个协议，我们使用一个自动染色机，但平等的结果可以通过手动染色获得。 </li></ol></li></ol> 3.现货在组织芯片（TMA手动工具包） <ol><li>创建一个组织的方向图，如2.1描述和分配点的所需位置（S）。 </li><li>填写TMA模具融化的石蜡在嵌入站。由于模具冷却，嵌入到底当场在TMA模具在由组织定向图所示所需位置的边缘（S）。根据需要，召开现场会（S）直立钳，直到石蜡公顷s凝固足够，现货（S）也不会摔倒（1-2分钟）。 </li><li>根据制造商的指示，并就在数组利润率（ 图2D）点（S）的位置组装TMA内核，并按照标准切片和染色协议。 注:请参见步骤2.6看到在这个协议中使用的切片和染色方法。 </li></ol>

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作者什么都没有透露。

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，件需要至少4.5-5毫米。以确保蜡的适当浸润，然而，件应不超过7 mm。当脱水染料片为蜡渗透，染料会稍微浸出件在罗威ř浓度的乙醇。浸出不会影响最终产品，斑点将仍然是强烈的染色和清晰可见。浸出可能会影响其它组织，虽然如此，组织切片不应在同一浴进行处理作为染料片，直到片已达到100％的乙醇。 当把这些点进入摆着块，确保所有其它组织的位置是将现场之前完成。由于现场被渗透与石蜡，收件人块熔化的蜡就可以开始融化当场核是否允许坐太久。先安排所有的组织块，加当场核心，并立即块转移到冷板，以防止斑点的任何熔化。 当场就可以进行修改，以适应大多数组织学实验室的工作流程。染料的颜色可以为每个应用最佳的对比度来改变。红色或黑色​​染料产生的免疫组化染色切片清晰的高对比度，W往往微不足道的红点是不一样的排列肾切片的H＆E染色的幻灯片醒目。对于H＆E幻灯片，绿色或黄色斑点往往会提供更高的对比度。此外，在过程中如热诱导抗原修复免疫组化进程高热，羟乙基琼脂糖融化。当场制备过程中的BSA加入到染料保持在即使经过高温检索方式滑动颜色鲜艳的斑点。如果滑动将不会暴露在高温，波塞可以省略更快点生产。 利用现货的最关键的环节是在创造和维护一个清晰，简明的方位图，并把现场当忠实遵守地图。如果使用得当，现货减少混乱的组织安排，并简化了技术人员和研究人员之间的交流，作为嵌入技术人员可以简单地表示点的位置和Tissu酒店的关系下载到它在地图上。 SPOT是所有步骤的幻灯片准备允许高度一致的部分安装在滑轨，便于下游分析清晰可见。与此相反，以生理标记，光斑在每节清晰可见并且不修改所述组织以任何方式，防止引入假象或梗阻形态的。不同于不对称嵌入组织的方法，当场让技术人员和研究人员组织的安排灵活性的显微镜下，以优化的可视化分析。在的TMA，当场就可以被放置在主阵列外，省去了空格，非对称组件，或信标核心，允许有价值的组织切片的整个阵列组成。当场也可用于指示解剖方向的组织样本（ 例如，远端或近端）。现货对组织样本的结构和过程中很容易和一致取向的简单和低成本的解决方案nalysis多组织块和组织芯片的。

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。组织取向图应包括与提供给研究者的幻灯片和详细解释所以会有在显微分析没有混淆。 图2A示出切片凝固，着色的琼脂糖凝胶塞的结果。 图2B示出嵌入的结果在切片琼脂糖凝胶塞S在石蜡块中。 图2C示出了使用真皮冲，以产生光点核的结果。 <img alt="图1" src="/files/ftp_upload/52868/52868fig1highres.jpg" width="700" /> 图1:现货芯很容易创建和组织块和幻灯片容易看到（A）点显然是在嵌入式模块可见。 （B）的斑点允许容易取向和组织鉴定含有来自不同个体/治疗组出现类似组织的多组织块。 （C）的斑点允许将要用于有价值的组织样品的组织微阵列的每一个核心。现货（D）显微镜查看邻近TMA核心。箭头指示的地方。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52868/52868fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </P&gt; <img alt="图2" src="/files/ftp_upload/52868/52868fig2highres.jpg" width="700" /> 图2:斑核的制备 （A） 的凝固羟乙基琼脂糖塞释放从Eppendorf管中并切成横向成5mm切片。 （B）的多片被嵌入在单个石蜡块和（C）一种皮肤冲头用来除去斑点芯。 （ 四）点嵌入到组织芯片的边缘。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52868/52868fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 ...

spot-the-correct-tissue-every-time-in-multi-tissue-blocks

Nanyang Technological University

Research

JoVE Journal

Biology

11.7K 次观看.  Frederick National Laboratory for Cancer Research. 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。 

视频: SpOT the Correct Tissue Every Time in Multi-tissue Blocks

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

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind 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

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

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 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

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

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

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

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

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

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

操纵小鼠泪腺

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

蛋白赖氨酸甲基转移使用SPOT肽阵列特异性分析

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

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

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 the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount 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 ...