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微升施用标记的化合物用于后续步骤的标准曲线的制剂。 注意:重要的是，标准从同一批次向动物给药标记的蛋白质的，以避免在荧光标记效率由于变性的差异进行。为了正确地去除背景fluores从后荧光测量萤光，未处理的动物必须使用相同的方法/试剂作为治疗的动物被处死。 </li><li>管理与使用任一尾静脉或股静脉（其通过1厘米的切口在腹股沟访问）先前描述1的IV注射。镇痛，如果制作腹股沟切口，辖卡洛芬皮下剂量为5毫克/公斤，并与伤口夹子闭合之前申请sensorcaine到切口部位。要通过鼻内（IN）航线管理，将动物在仰卧位。 </li><li>使用2μL移液器，制定标记化合物的1μL下降。简言之，将动物的头从麻醉衣领滑动以允许访问鼻孔。 </li><li>吸移管的尖端放置到单个鼻孔的开口，慢慢压下柱塞，从而允许所述液滴向下运行的鼻孔进入鼻腔。重复每1分钟，交替鼻孔每一滴直到全部剂量广告服侍。 注意:通过在管理的最大音量不得超过10 - 15μL鼠。 </li><li>他们每移动到一个单独的笼子前，留在仰卧位动物麻醉下进行10分钟的最后给药后。 注意:不要离开无人看管的动物，直到他们重新获得足够的意识，以保持胸骨斜卧。 </li><li>给药后1小时，如之前重新麻醉的动物，并使用PBS（210.0毫克/升KH 2 PO 4，9,000.0毫克/升氯化钠，726毫克/升的 Na 2 HPO 4·7H 2 O）在经由穿心灌注牺牲他们10毫升/分钟，总共20毫升。 注意:动物在处死灌注以从组织中移除所有的血液需要确定的标记的化合物的血管外的浓度。灌注试剂可导致改变的背景荧光，并应保持一致。牺牲的方法应该是一致的内的组。牺牲的不同方法会导致在脉管内变化的血液水平，改变了组织的背景荧光。 </li></ol></li><li>组织集合<ol><li>如上述用于漂洗除去组织描述制备PBS中。 注意:如果另一个缓冲器被用作灌注液，在相同的缓冲液冲洗组织。 </li><li>通过简单的夹层中取出心脏，肺，胃，肝，脾，肾。在PBS溶液清洗使用实验室之前湿巾抹干或吸走多余的水分，并把组织上的成像垫子。 </li><li>斩首鼠标和使用刀具骨或骨钳去除头骨的顶部。使用镊子，小心地取出大脑，保持嗅球完好。冲洗，干燥，并将其放置在所提供的影像垫。 </li><li>要通过快速弹射7去除脊髓，填充10毫升的注射器，用PBS和附加迟钝18号针头。 注:18号适合许多鼠标线的成人椎管。所需的直径将动物大小而异。 </li><li>置于俯卧位的动物，以确保椎管在断头站点的开口被干净地切割和自由的血液。 </li><li>来自各地的脊柱去除皮肤。使用锋利的刀骨或剪刀，通过在腰节直接喙臀部脊柱切断。 </li><li>简单地说，用PBS冲洗可视化椎管。轻轻插入针头进入椎管。 注:针应该感到舒适，并可能需要一些缓慢滚动的插入过程中来回穿梭。如果针松的感觉，更切喙可作出或可能需要较大的直径针。 </li><li>用拇指和食指，将压力施加到插入针周围的脊柱。按下中度和恒压活塞。脊髓将弹出完整出喙开放的脊髓柱。 注:彻底清除，冲洗，以及所有主要器官可以在5合理完成烘干 - 7分钟。这样短的时间内阻止对PBS扩展存储，这可能会导致过度曝光，并从组织标记的蛋白质浸出的需要。 </li></ol></li></ol> 2.全器官离体荧光成像<ol><li>成像参数<ol><li>启动图像采集的软件。在收购控制面板的右下方，点击"初始化"。 </li><li>在"成像模式"，选择"荧光"复选框。在"曝光时间"，选择"自动"。 </li><li>在"分级"，选择"小"和F /停止设置为2。 </li><li>选择535 nm激发和580 nm发射过滤器。 注意:这将取决于所使用的标签上会有所不同。 </li><li>选择"视图B场"，并设置圆盾ŧ高度0.3厘米 注意:这将取决于组织被成像而变化。 </li><li>一旦温度指示器变为绿色（意味着机器完全初始化），将组织到提供黑色背景垫，从而确保组织被完全彼此分离。放置在成像器的垫，以确保所有的组织都在所选区域的网格内。较大的和非均质的组织，如全肝，应布置，以便减小波瓣重叠。 注意:组织可以一起在单个组被布置，使一个图像，以容纳一单个动物或相似类型的所有组织中。这使得更容易稍后的分析和组织。如果在荧光强度大的差异是各种组织中本，最好是图像它们单独或作为同一组织类型的基团，作为一个单一的曝光时间可能不适合于所有的组织的强度。 </li><li>点击"获取"。 </li><li>成像后，立即取出组织并存储或冻结它们定量组织学，如在步骤4中概述。 </li></ol></li></ol> 3.标准和图像处理<ol><li>标准<ol><li>在PBS中，创建在动物用于治疗，开始于原始浓度各蛋白质的两倍连续稀释，以总共15稀释液。 注意:浓度范围应该旨在包括预期的组织浓度，并可能需要与原始样品的附加的稀释，这取决于起始浓度。 </li><li>吸取100μL的每个标准成黑色的96孔板的每个孔中的。包括纯PBS中的一个孔中，以获得背景荧光的测量。 </li><li>图像与用于组织成像相同的参数标准。 </li><li>在图像采集软件"工具选项板"下的"投资回报工具"，选择"感兴趣区域（ROI）"圈子，并绘制一个井周围的投资回报率。复制ROI所有其他填充井，然后点击"衡量投资回报率。" </li><li>减去在从其他的标准值的纯PBS中平均辐射效率的值;这消除了背景荧光。上绘制散点图对已知浓度值。 注意:得到的线性趋势线的方程可以被用于计算相对荧光值。 </li></ol></li><li>图像处理<ol><li>在图像采集软件，选择自由ROI工具，绘制的ROI周围每个独立的组织。点击"措施"。 </li><li>用所得平均辐射效率的测量，减去未处理动物的每个组织类型测量的值和先前创建标准曲线上绘制这些值。 注意:得出的相对荧光值提供的快速，准确的快照标记化合物的生物分布。 </li></ol></li></ol> 4.定量组织组织学<ol><li>对切​​片的制备和冷冻的组织<ol><li>创建或购买足够的尺寸以冻结所选组织和低温恒温器的容器中使用。周围适当尺寸和形状的物体包裹铝箔效果很好。 </li><li>通过填充一个500ml的烧杯中的一半用干冰和加入异丙醇的〜350毫升，以便有足够的液体的干冰以上以部分地浸没冷冻容器准备异丙醇和干冰的混合物中。 注意:液氮经常导致最佳切割温度化合物（OCT）和包埋组织的断裂，应该避免。 </li><li>与华侨城部分填充冷冻集装箱，确保有不存在泡沫。如果有气泡，使用镊子工作气泡从溶液或类似的工具。 </li><li>除去组织从PBS中并干燥它彻底，或者用一个实验室擦拭或通过触摸细腻组织到实验室的边缘擦拭和允许液体吸走轻轻拍打它。 </li><li>轻轻地放在组织到华侨城的冷冻集装箱内，确保不引入气泡组织下方。一旦组织就位，添加OCT中直至组织被完全覆盖。 </li><li>浸没冷冻集装箱尽可能是可能的，而不使异丙醇进来与暴露十月接触的容器内。允许30 - 90秒（或可能更多更大的组织）的OCT和组织在-80℃保存，只要需要之前完全冻结。 </li></ol></li><li>标准的制备切片。 <ol><li>准备并通过去除筒的端部正确标记1毫升一次性使用的注射器，使得所述注射器的整个长度为一个直径。这将允许冷冻汽缸后面将要推出。 </li><li>我们ING OCT中，创建施用的化合物的15步两倍系列稀释，如用PBS先前完成的，但用1 mL的最终所得的体积。包括用于去除背景荧光值纯OCT的一个样本。 </li><li> OCT的粘度使得充分混合困难。反转试管，华侨城再次反转之前，彻底解决。重复此10 - 20次，以确保一均匀的混合物。混合过程中，使标准溶液在4℃和避光。 </li><li>混合后，再次准备异丙醇和干冰的混合物中，如前。 注意:液态氮也可以代替异丙醇/干冰混合物的使用，只要它们冻结固体后注射器迅速取出。 </li><li>绘制OCT标准入注射器，同时注意引进尽可能少的气泡越好。拟定OCT溶液后，立即直接浸注射器插入异丙醇，并允许该溶液完全冻结20秒 - 注射器10中。存储注射器在-80℃下，只要必要的。 </li></ol></li><li>组织和标准的切割<ol><li>将两种组织和标准-20°C和足够的时间让所有样品来温度。 注:理想的切削温度一般落在周围的-10°C范围内对大多数组织中，但它最终取决于组织的大小和脂肪含量必须凭经验细化。 </li><li>使用低温恒温器，切割组织成20微米厚的切片，并将其安装到滑动，如前所述8。在-20℃保存幻灯片和切割过程中，直到扫描使他们免受光线尽可能远。 注意:重要的是在这里，要测量的部分在整个组织的整个厚度被适当地分布。 </li><li>要安装到标准的低温恒温器吸盘，采取从冷冻注射器和温暖外3 - 5秒通过霍尔鼎在手里。推动柱塞，直到在OCT气缸的短节（约1厘米）是注射器的外部。 <ol><li>用剃刀刃，切通过气缸和垂直放置所得汽缸部上的卡盘，使用OCT以保持它在适当位置。这种构造应该导致良好定义的圆圈被切断。 </li></ol></li><li>切片气缸20微米，从每个稀释放置一个片到单个滑动，使一个滑动表示整个标准曲线。储存在-20℃下将载玻片直到准备扫描。 </li></ol></li><li>扫描和定量<ol><li>启动扫描阵列软件，点击"543激光"开始激光启动过程。等待15分钟的激光做好准备。 </li><li>一旦激光准备就绪时，从-20取出幻灯片℃，并将它们移动到一个大的，光保护的容器来的温度和干燥3分钟。这将防止受潮在SLI德扫描仪。 </li><li>加载滑入上滑动扫描器滑动容器。点击"扫描"，选择"运行轻松扫描。" </li><li>设置扫描分辨率为50微米。 </li><li>在"荧光团"选择第一个"使用"复选框，并选择"TRITC"设置PMT增益为80％。 注意:这些设置将取决于荧光团和标记分子的浓度变化。 </li><li>在左手侧，确保了扫描区域包括整个幻灯片。点击"开始"。 </li><li>扫描后，选择"文件"，"另存为"，然后将图像保存为.tif。 注意:组织和标准的幻灯片应在相同的设置进行扫描。 </li><li>加载生成的图像转换成ImageJ的，选择投资回报组织和标准各地。在"分析"选项卡中，选择"设置测量"，选择"平均灰度值。"点击220;措施"。 注意:与前面的技术中，背景荧光必须从所有测量值中减去。 </li><li>从绘制对已知浓度的标准平均灰度值测量，以创建标准曲线。 注意:从身体组织将所得的测量应在每个组织内进行平均并绘制该标准曲线上，以确定在组织内的浓度。 </li></ol></li></ol>

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GLB is owner of Leflore Technologies, LLC, a private company working to develop ELP-based drug delivery technology. Leflore Technologies provided no support for the publication of this paper or for the generation of data within it.

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图...

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 ...

the-use-ex-vivo-whole-organ-imaging-quantitative-tissue-histology-to

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

9.5K 次观看.  The University of Mississippi Medical Center.  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。 

视频: 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

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

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

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

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

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

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

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 ...

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

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

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

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

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

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

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

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

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

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations 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, ...

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

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

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

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

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 ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

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

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

用于荧光标记大促卵体定量的自动成像和分析

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

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肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

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小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

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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

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Adipo: 三维脂肪组织成像的组织清除方法

用于荧光标记大促卵体定量的自动成像和分析

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