我们感谢他们正在进行的合作A. Tager和B。乳木果，并提供用于演示目的的肺组织。这项工作是由国家卫生补助HL - 092961研究院的支持。这项工作是在纳米系统（CNS），国家纳米技术基础设施网络的成员，这是由国家科学基金会的支持下国家科学基金会奖ECS - 0335765中心部分。中枢神经系统是在哈佛大学艺术和科学学院的一部分。

1。肺组织地带准备<ol><li>肺组织是高度兼容，难以切成条状原子力显微镜表征。瞬时稳定切割肺结构，膨胀小鼠离体肺，气管，50毫升/公斤体重2％的低凝点琼脂糖（用PBS配制）加热到37℃铁气管和冷却在4浴的PBS膨胀肺° 60分钟的彗星。琼脂糖凝胶和变硬空域轻轻稳定在这个区间5的肺结构。琼脂糖浓度较高，即3-4％，可用于进一步加强组织切割稳定。 </li><li>成5 × 5毫米的长度和宽度和厚度在400微米条用剃刀或解剖刀刀片剪切琼脂糖稳定的小鼠肺组织，然后洗一个PBS水浴预热至37条℃用100毫升5分钟以去除残留的琼脂糖台式加热搅拌盘上的玻璃烧杯。为了排除大的航空公司和船舶，切条从遥远从主茎支气管胸膜地区。如果航空公司和大血管进行成像，切条近端主茎支气管肺组织。 </li></ol> 2。的原子力显微镜Microindentation和荧光成像<ol><li>为了隔离感兴趣的领域的AFM microindentation，组织可以进行可视化相衬显微镜或免疫染色，荧光显微镜观察。免疫，组织带块与5％，从2小时或在室温下没有固定通透源所需的二次抗体（PBS）的血清。 </li><li>与相应的抗体孵育组织地带（如兔抗Ⅰ型胶原的可视化间质胶原（1:250稀释），兔抗层粘连蛋白（1:250稀释）的可视化基底膜）在12孔板以及中在1小时轻轻摇动摇杆，洗5分钟，用PBS样品的3倍，其次是适当的二级抗体结合荧光标记（如的Alexa Fluor 546山羊抗兔二抗（1:250稀释））1在室温小时。 </li><li>广泛组织带用PBS和存储在PBS清洗4 ° C </li><li>紧接原子力显微镜表征前，将组织地带到15毫米的聚- L -赖氨酸包盖玻片盖玻片解除低于浮动组织，确保组织地带盖玻片表面均匀传播，如有必要，三明治的地带第二清洁，无涂层的盖玻片和应用温和的压力，以协助组织附着的聚- L -赖氨酸包被的盖玻片。 </li><li>每一轮microindentation实验前制造的指令立即校准AFM系统。确定两个关键参数:1）悬臂弹簧常数使用热空气中的6的波动方法;，2）悬臂偏转灵敏度，这是用于规模的光电二极管的输出信号，以实际悬臂偏转距离参数，δD 。获得一个干净的载玻片在PBS一个标准的力 - 位移曲线，然后计算出的力 - 位移曲线（图1B）的斜率校准偏转灵敏度。力 - 位移曲线测量，延长对针尖和样品表面在一个位置（不包括在XY方向光栅）缩回，悬臂的偏转，δD尖位移，ΔZ功能监测， 。 </li></ol>我们建议使用一个直径为5微米的硼硅球形提示（Novascan）氮化硅三角形悬臂。使用AFM探针弹簧常数为0.06牛顿/米，我们有机械的特点与软质材料剪切模量跨越100至50,000霸。 <ol start="6"><li>样品盖玻片用纸巾擦拭底部表面，固定到一个真空润滑脂的标准载玻片，安装在载玻片上的原子力显微镜样品台上，和封面用500μlPBS（室温）的组织。 </li><li>设置显微镜目镜观看对齐针尖在视野的中心，通过调整显微镜样品台上。选择组织的利益方面，通过移动的原子力显微镜样品台上，然后切换到CCD摄像头，查看记录相衬图像和/或组织的荧光图像，根据需要。 </li><li>移动针尖慢慢向下，直到它与样品接触的是执行标准的AFM参与操作。 </li><li>精确的原子力显微镜表征microindentation软样品需要小压痕深度，以避免大的地方株无效赫兹的弹性模量计算模型。要避免大的菌株，进行触发模式设置悬臂最大挠度（触发点）到500 nm压痕。这偏转限制将抑制最大压痕力小于30 NN（F =悬臂弹簧常数*位移，或F MAX = 0.06 NN / NM * 500纳米= 30 nN个）。 </li></ol>应选择压痕速度足够慢的探索弹性而软的样品 7粘弹性能。一个2-20微米每秒的速度范围内是适用于肺组织。 <ol start="10"><li>对于感兴趣的领域从单一测量，原子力显微镜的探针移动感兴趣的位置，并执行一个单一的缩进收集一个标准的力 - 位移曲线（XY扫描探头，只有在Z轴方向移动没有）。 </li><li>自动映射一个感兴趣的区域，切换到部队的地图模式，选定的区域内，选择扫描尺寸和采样点。在部队的地图模式，整个样品表面之间的缩进运动针尖栅格，并在每个点的范围内收集个人力 - 位移曲线，一个定义的采样网格。更多的采样点，将提供更高的空间分辨率，但延长的映射所需的时间。我们发现它实际使用了一个16 x 16个样品的网格地图缩进速度在每秒20微米，可以在大约10分钟完成一个80 × 80微米的区域。 </li></ol> 3。 AFM数据提取<ol><li>要计算的杨氏模量，适应力-位移曲线赫兹的球形压痕模型，利用最小二乘拟合非线性弯曲 8（图1，C ）: <img src="/files/ftp_upload/2911/2911eq1.jpg" alt="公式1" /> 其中F = k 彗星 *Δ 剐 ，的力量来弯曲的悬臂，K C是的悬臂弹簧常数，ř是领域的尖端半径，δ=Δž - Δð是缩进和υ是样品的泊松比率（υ= 0.4肺组织9）。 </li><li>为了评估适合的质量，计算出的SSR值，或数据和拟合值之间的差异的平方的总和，在非线性曲线拟合。消除"坏"的曲线，与大SSR值丢弃的数据不可靠或联合国解释测量。 </li><li>如果需要，弹性模量（E）转换为剪切模量（G），使用关系:E = 2 ×（1 +υ）× G.要可视化的空间刚度力的地图模式中收集的图案，等高线图图模量数据（例如，在16 × 16的样本电网覆盖一个80 × 80微米的面积）。 </li><li>要处理大量的力曲线数据，可能会被写入一个自定义的算法自动适应力 - 位移曲线，提取模量，和/或使用相同的程序和参数，在3.2和3.3（如MATLAB）中所述的情节elastographs。 </li></ol><img src="/files/ftp_upload/2911/2911fig1.jpg" alt="图1" /> 图1（a）使用球形探头（Dimitriadis大肠杆菌等2002生物物理学J 8许可转载）一个刚性基板上的软样品的原子力显微镜microindentation的原理图。 （b）代表力 - 位移曲线，收集在PBS洁净的载玻片，以确定悬臂偏转灵敏度。 （c）代表力 - 位移曲线从软（蓝色）和僵硬的（红色），显示相应的参数（一）样品收集。 4。代表性的成果: 图2A显示了肺实质带附着在涂层聚- L -赖氨酸的盖玻片在PBS代替上述地区的利益直接与原子力显微镜的探针和准备microindentation映射。图2b显示，在适当的剪裁和染色的组织，肺实质的肺泡微架构是保存完好，没有固定或通透的组成部分基底膜层粘连蛋白的免疫荧光染色观察。图2 CD显示我在收获以前，用PBS（图2C）处理的小鼠，或与博莱霉素（图2D）处理，诱导间质纤维化 10新鲜不固定的肺胶原蛋白的免疫荧光染色。 图3a显示样品的力 - 位移，从原子力显​​微镜microindentation获得的地块。随着同样适用于力量，针尖会产生一个大的缩进于软的区域（蓝线），在一个相对的"扁平化"的一个小的压痕和地区的围追堵截"陡峭"的力 - 位移曲线与力 - 位移曲线（红线）。为了获得清洁力曲线，每个缩进后针尖需要完全缩回从样品的表面和接触之前，未来的缩进。这种接触的自由状态，对应于图3A，提示没有悬臂偏转转换曲线的平坦区域。图3B显示了一个典型的虚假曲线不平坦的地区，发生笔尖的时候，不能完全缩回从样品表面（如软样品ttaches到TIP）使其无法确定接触点。如果提示是完全被困在软样品，曲线看起来可能如图3C所示，没有明确的偏转，但噪音小。 图4显示了剪切模量提取的数据空间作为刚度地图（elastographs），剪切模量的色阶显示力 - 位移曲线。 Elastographs表现出显着的差异，在正常的范围和组织剪切模量的分布（图4A）和纤维化（图4B），特别是在肺纤维化样品肺实质和剪切模量较大的空间变化，。 <img src="/files/ftp_upload/2911/2911fig2.jpg" alt="图2" /> 图2:（一）相衬显微鼠标肺实质条显示了原子力显微镜的探针。比例尺，50微米。 （二）在正常小鼠肺组织层粘连蛋白的免疫染色显示正常肺组织的肺泡微架构。白框表示典型的由80 -80μm的弹性映射区域。比例尺:20微米。 （C和D）我在生理盐水（C）和博莱霉素（四）治疗小鼠肺实质的胶原蛋白免疫染色。比例尺，100微米。 <img src="/files/ftp_upload/2911/2911fig3.jpg" alt="图3" /> 图3（一）代表解释力-位移曲线，分别收集液（蓝色）和博莱霉素治疗（红色）小鼠肺实质。黑线是从球形赫兹模型的最佳契合，并计算其相应的杨氏模量的标记。 （B，C）代表联合国解释力 - 位移曲线。 <img src="/files/ftp_upload/2911/2911fig4.jpg" alt="图4" /> 图4。生理盐水（A）和博莱霉素治疗（二）小鼠肺组织收集的代表elastographs 。彩条显示在千帕的剪切模量。轴标签显示的空间尺度在微米。比例尺:20微米

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没有利益冲突的声明。

肺组织使用原子力显微镜microindentation的机械特性，提供了前所未有的空间分辨率（图4），提供一个独特的视角对微观组织的刚度变化。由于其效用的一个例子，以前的宏观尺度的测量表明在正常和纤维化的肺组织条近似倒电容与纤维化11,12增加2 3倍。相比之下，原子力显微镜microindentation显示，一些地区参展〜30倍，在正常肺组织10中观察到的中位数以上的剪切模量的增加，该组织变硬是高度本地化的。作为基体刚度是目前已知看房影响细胞功能，这些地方的测量提供了宝贵的参数，以提高细胞肺癌细胞培养研究biofidelity。 几个实际问题的产生与使用细条状的肺组织。条表面完全平坦，为组织配置文件如下的基本肺泡架构。 AFM系统自动调整在压痕探针在Z方向的位置时，样品表面的高度变化超过15微米的小，以帮助克服这一挑战。测量是在室温，37℃，所以不能在这种温度变化所造成的机械性能偏差评估，尽管他们会预计将轻微。底层的肺泡壁结构上观察到的机械性能的影响是难以确定，与目前的光学显微镜设置。举例来说，将是可取的，以确定是否肺泡壁时，表现出的各向异性和不同的力学性能符合或横墙的平面的方向缩进。然而，当前的标本厚成像系统没有3D功能，因此它是不可能在每个接触点，以确定当地的肺泡壁方向。最后，细胞成分的测量机械性能的影响仍有待完全阐明。在这里详细的方法，没有作出努力，特别是消除组织的细胞成分。然而，细胞表面压痕可用目前是不大可能是可行的，自组织收获和切割组织进入到肺泡的必要性所经过的时间。具体的实验，去除细胞，或与活细胞重新填充矩阵，评估组织刚度产生的变化会出现必要的。 由于这些测量需要新鲜不固定的组织，从组织收获已过测量时间应尽量减少和样品应保存在4 ° C，以避免在机械性能的变化。每当组织条在洗涤或染色，以便产生最小的失真或损坏容器之间转移支付，应特别注意。原子力显微镜在液体中的应用，一个关键步骤是削减尽可能扁平的组织和支持盖玻片固定的样品。如果可用，作为vibratome或组织切片机自动切片机，可以用来切割高度厚度均匀的片。重要的是立即组织带附加AFM测量前，尽量减少AFM测量经过的时间，作为样品将最终脱离的盖玻片。一个有用的观察，出现较大条连接更加稳定盖玻片，并留在原地，持续时间较长的PBS比较小条。 原子力显微镜的microindentation可以表征样本，涵盖了广泛的范围从100帕到50千帕（剪切模量）一起使用时，一个直径5微米的球形提示一个标准的0.06牛顿/米的悬臂。这个范围可以扩大使用与不同的弹簧常数探针;原子力显微镜探针与球面玻璃从0.6至12微米范围从0.01至0.58牛顿/米的直径和弹簧常数不等市售（例如Novascan），常用3的提示。 5微米的球形提示，提示和组织之间的理论的接触面积约5-9微米为400-700纳米压痕（ 图 1A ）2。更大或更小的技巧可以被用来提供更大或更小尺度的空间分辨率。锥体提示也被用于在原子力显微镜microindentation 13-16，提供更小的接触面积，从而增加映射的空间分辨率，虽然数据拟合这个尖几何形状更为复杂。 这种方法应注意的几个限制。肺历来被机械的非侵入性的特点，例如使用压力-容积分析 17或整个肺部穿孔缩进 19,20 。这里描述的改变肺结构的重要途径之一通过空气液体INT的损失，如侵入性的方法erface，通常在充满空气的肺和预应力的损失，保持呼吸道肌肉放松后肺局部通胀存在。这些限制是在肺组织带18的所有测量的共同。然而，值得注意的是，中位数刚度测量在正常肺组织的实质（剪切模量〜0.5kPa）不大幅不同于基于 19,20休息卷完好肺部穿孔缩进的估计。虽然肺组织表现出越来越变形的非线性劲，它是不可能的，在严格的方式进行测试，这个属性是否仍然存在，这里的方法，以微观尺度。赫兹模型假设样本的同质性。但是，大多数生物材料，包括肺实质，越来越多地在减少空间尺度上的异构。样本的异质性，可能会导致文物如根据压痕深度，即取决于层或元件，是变形的杨氏模量的变化。在XY平面的异质性，可以通过仔细选择合适的球形针尖大小，取决于Dimitriadis EK等提出的生物材料的微观结构有限8这是更难以预测或不正确的赫兹模式错误，由于材料。在z方向的异质性。 Azeloglu等。最近提出了一种混合计算模型来表征异构基板的弹性性能与离散嵌入式夹杂21。他们的新技术提供了潜在的手段来克服赫兹分析的局限性的非均质材料计算列入属性。 赫兹模型还假定绝对的弹性行为，而生物材料通常显示随时间变化的粘弹性行为。一个完整的组织粘弹特性，可通过改变所使用的压痕速度。更重要的是，以前的宏观尺度的机械测试显示正常和纤维化的肺组织肺组织的力学性能弱的频率的依赖，并保存所有频率的正常和纤维化组织的力学性能之间的差异测试11。这些研究结果有力地表明，使用一个单一的压痕速度与原子力显微镜的机械性能的测量捕获组织的力学性能的变化，伴随着肝纤维化的一个重要方面。 0.4肺组织在这项工作中使用的泊松比是从宏观的测量9。不幸的是，在微观尺度的泊松比，并根据病情的任何变化都没有在文献中。 22作为替代品E，E /（1 -υ2）或（1 -υ2）/πE（表示弹性常数k）可以计算出原子力显微镜microindentation和报告时的泊松比是未知的。对于大多数生物材料的泊松比是在0.4至0.5的范围内，由于其含水量高。在0.3-0.5的范围内，只有从1.10-1.33因子1 /（1 -υ2）不同，这样合理的泊松比的变化对报告模量的影响有限。 ，我们为纤维组织相对正常组织报告的剪切模量的增加幅度的数倍，这意味着与泊松比的变化相关联的错误是轻微的相对力学性能的变化观察。 具体应用条件和后续力 - 位移曲线的不同人群的特点是受力 - 位移数据的分析，可以使用实际的算法和代码。如果感兴趣的是更复杂的分析，一个可咨询林等人的工作。23 。作者编译成一系列的协同战略的一个算法，克服了许多先前已阻碍了努力自动化赫兹压痕数据模型的拟合并发症。 其他几个领域的进一步发展，并利用此方法可用。其中一个是在可视化没有抗体标记的肺泡壁有兴趣的情况下，弹性蛋白和胶原蛋白可以从他们在绿色光谱autofluorescent信号的可视化。另一方面，更好的成像，使用或者稀释剂的组织切片，三维成像技术，或两者，可以加强相互联系的能力与组织架构的基本力学性能。虽然目前的方法使细胞外基质成分，如胶原蛋白和层粘连蛋白的染色和可视化，更多的努力可以针对染色细胞表面标志确定具体的细胞群，并在这些人群中附近机械的微环境特征。 Alt键ernatively，组织可收获小鼠表达谱系标记或细胞的特定蛋白质荧光标记的追求相同的目标。最后，该方法的详细，出现在这里非常适合描述其他解剖特点，如改造中高血压的船只，并改造在哮喘气道，肺，。根据其目前的发展和进一步提高的潜力状态，原子力显微镜microindentation似乎准备组织刚度的变化，陪在肺部疾病进展产生有价值的见解，无疑将是有价值的，在时空变化特征在刚度的各种其他软组织。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>针尖</td><td> Novascan </td><td> PT.GS </td><td> 5微米的高硼硅珠，0.06牛顿/米</td></tr><tr><td>聚- L -赖氨酸盖玻片</td><td> VWR </td><td> 354085 </td><td>屋宇署BioCoat 12毫米的圆形第一玻璃</td></tr><tr><td>琼脂糖凝胶温度低</td><td>西格玛</td><td> A0701 </td><td></td></tr><tr><td>兔抗- I型胶原（Rb的PAB）抗体</td><td> Abcam公司</td><td> ab34710 - 100 </td><td></td></tr><tr><td>的Alexa Fluor 546山羊抗兔抗体</td><td> Invitrogen公司</td><td> A11010 </td><td></td></tr><tr><td>兔抗-层粘连蛋白</td><td>西格玛</td><td> L9393 </td><td></td></tr></tbody></table>

micro-mechanical characterization of lung tissue using atomic force microscopy

矩阵刚度强烈地影响着1-3的贴壁细胞生长，分化和功能。在宏观尺度的组织和器官在人体内的刚度，跨越几个数量级4订单。更别提了解如何刚度空间内组织的变化，刚度变化的范围和空间尺度疾病过程中，在组织重构的结果。为了更好地了解如何在矩阵刚度的变化在健康和疾病的细胞生理学，在相关的空间尺度，居民细胞获得组织刚度​​的测量是必要的。这是肺，高度兼容和弹性纤维组织基质重塑是一个突出的特点，如哮喘，肺气肿，高血压和纤维化等疾病尤其如此。为了描述在空间尺度上，有关居民细胞肺实质本地的力学环境，我们已经开发出直接测量新鲜小鼠肺组织利用原子力显微镜（AFM）microindentation局部弹性性能的方法。有了适当的选择，AFM的压头，悬臂式，并压痕深度，这些方法允许相衬和感兴趣的区域的荧光成像并行局部组织的剪切模量的测量。组织带系统抽样提供了组织机械性能，剪切模量，揭示了当地的空间变化的地图。机械性能及相关的解剖和病理特征之间的相关性说明如何刚度与基质沉积在肝纤维化的变化。这些方法可以扩展到其它软组织疾病的进程，揭示局部组织的力学性能如何跨越空间和疾病的进展而异。

Watch this Scientific Journal Video about Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy at JoVE.com

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

细胞外基质的刚度强烈地影响着贴壁细胞的多种行为。矩阵刚度不同空间在整个组织，并在各种疾病的情况下发生的修改。在这里，我们开发方法来表征的空间变化，在正常和纤维化小鼠肺组织的刚度，使用原子力显微镜microindentation。

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

micro-mechanical-characterization-lung-tissue-using-atomic-force

Research

JoVE Journal

Biology

22.8K 次观看.  Harvard School of Public Health. 细胞外基质的刚度强烈地影响着贴壁细胞的多种行为。矩阵刚度不同空间在整个组织，并在各种疾病的情况下发生的修改。在这里，我们开发方法来表征的空间变化，在正常和纤维化小鼠肺组织的刚度，使用原子力显微镜microindentation。

视频: Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

科研

生物学

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

免疫荧光法Barrett食管细胞的表征

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

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

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

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

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus 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 ...

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. 
This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. 
Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

使用连续截面电子显微镜对肝细胞中组织间接触位点进行三维表征

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is ...