作者实验室的研究是由美国国立卫生赠款R37 - HL041026，R01 - HL086483和R01 - HL056786（SSS）和F32 - HL097463和T32 - AR048523由美国公共卫生服务（PB）研究院的支持。

1。鼠标板，肌肉底座，身体的楔形和灌流解决方案<ol><li>鼠板:一个长方形的透明有机玻璃板（6"宽x 8"长X 3 / 16"厚）适合在显微镜的阶段。"鼠板"是麻醉鼠标固定在手术准备提睾肌。 </li><li>肌肉基座:一个透明的硅胶橡胶底座Sylgard 184，这是根据制造商° S指示的混合的准备。一个方便的模具是15毫米厚x 50毫米直径的直径一次性培养皿。 Sylgard块切割成所需的形状，用刀片（见图1c）。。一个清晰的防水硅酮胶薄薄的一层是用于安全Sylgard块鼠板。有用的提示:在一个小时的真空室脱气后浇入培养皿Sylgard，消除气泡和提高清晰度。以下脱气，固化为Sylgard时间缩短为50 ° C数小时的菜放置在实验室烘箱。 </li><li>楔形车身和加热平台:一个楔子（2"× 4"× 15 °），下面的鼠标和定位对基座倾斜身体前倾，有利于延长从它的起源底座，提睾肌。结合在表面上的铝制平台，提供导热。该平台是加热电阻连接到直流电源（图1A）。校正平台温度〜40℃食管温度保持在〜37 ° C。否则，从一盏灯的辐射热。 </li><li>销。为了确保提睾肌的底座上，别针准备从0.15毫米的昆虫引脚已进入一个"L"形弯曲。 </li><li>生理盐溶液（PSS）。准备工作正在灌流（灌溉）准备在超纯水（18.2MΩ）H 2 O的碳酸氢盐缓冲PSS的不断股票的解决方案是准备在20X最终工作浓度和消毒，通过一个0.2微米的过滤器。好几个星期，当股票的解决方案仍然保存在4 ° C。准备分开的碳酸氢盐和盐混合与稀释实验的一天，他们阻止的碳酸氢钙沉淀。该公司股价由盐溶液（mmol / L的）:2638氯化钠，氯化钾94，40 MgS0 4，23.4氯化钙 。股票碳酸氢钠碳酸氢钠3 360。在当天的实验，都带来了各自的解决方案容量瓶终体积（通常为2升）对于一个给定的实验〜37 ° C水浴中放置非平衡的PSS与5％的CO 2 / 95〜15分钟使用气体分散％N为2调节pH值至7.4〜和防止沉淀。用5％的CO 2 / 95％，在整个实验二氮的PSS是不断冒泡。最后的工作液成分（mmol / L的）:132氯化钠，4.7氯化钾，1.2 MgS0 4，2，2氯化钙18 碳酸氢钠 3 。 </li></ol> 2。麻醉和手术的准备<ol><li>从体制上的动物护理和使用委员会的批准后，至少12周龄雄性小鼠。鼠标与戊巴比妥钠（60毫克/公斤）经腹腔（IP）注射麻醉。限制管确保鼠标在最初的注射证明是有用的。整个手术过程中和实验协议，麻醉维持补充剂（10-20％初始注射，IP）（每次30-60分钟;表示戒断反应到脚趾或尾巴捏）。提示:稀释苯巴比妥10毫克/毫升无菌生理盐水，注射前减少了过量的可能性。 </li><li>麻醉会影响体温调节，因此鼠标必须保持第一次注射后立即热烈。鼠标放置在一个金属载体上一个加热板的顶部（通风铝篮）（校准〜37 ° C）的效果很好。另外，热灯可用于照顾在一个适当的距离从鼠标位置。鼠标应监测每隔5-10分钟，直到适当的麻醉水平达到（缺乏撤退到脚趾或尾巴捏）。 15-20分钟后，可能需要的补充剂量。这是最好的，要耐心谨慎行事，以避免过量，特别是与肥胖或年龄的动物，。 </li><li>从下腹部，腰部，阴囊和腿通过仔细剃各自区域的头发被删除。应特别注意采取阴囊和睾丸，否则会损害提睾肌，避免创伤。将松散的头发，用一个新的酒精棉签。 </li><li>如果膀胱满了，会觉得像小葡萄经腹壁。排空膀胱，使用温和的压力，以防止鼠标，从小便在实验过程中睾准备。一个Kimwipe效果海绵，以收集T的尿液。 </li><li>鼠标在它的后面的位置，就趴在楔形，与它的腿跨越底座。每只脚绑一个丝线缝合（4-0或5-0）提供了一个系绳确保其对底座的裆鼠标。放置在胸前松散地固定楔形的磁带，保持整体车身的位置。通过使用显微切割剪刀和直角钳体视显微镜观察时，手术程序进行。 </li></ol> 3。开放提睾肌的手术准备<ol><li>阴囊的顶点（左边或右边）通过一个引脚放置和固定在底座放置在皮肤上的紧张局势。 PSS（34-35℃）灌流手术领域发起和维持整个清扫，保持暴露组织的温暖和潮湿。灯芯（从Kimwipe）的一端，旁边的阴囊和其他真空线定位，消除了污水PSS的。一个坚持有机玻璃板，并完全包围楔形底座作为"护城河"，以收集任何不送气的PSS的服务，作为一种有效的预防措施防止泄漏到显微镜的PSS的硅酮密封胶珠。 </li><li>切口是沿阴囊腹面。如果睾丸一直缩回进入腹腔提睾肌，小腹温柔的压力指示到国资委的睾丸。提睾肌的表面覆睾丸应确定在这个时候。小心取出摆脱提睾肌周围组织之间的阴囊皮肤和提睾肌的结缔组织。 </li><li>阴囊皮肤轻轻地缩回背后底座近端边缘和用大头针两侧固定。提睾肌表面的外表面，然后清除结缔组织。连续灌流期间清扫水合物的结缔组织，促进知名度和清除。 </li><li>通过提睾肌的顶点针是用来放置纵向张力下。一纵形切口，通过采取非常谨慎，以尽量减少血管供应中断的肌肉腹面的。这个组织损伤14准备外围附近的血流动力学改变。因此，最大限度地减少这些影响是用于数据的收集，编制中心附近的血管成像和生理研究。 </li><li>提睾肌是连接由一个薄的韧带下方睾丸附睾。一个侧面反映睾丸暴露此韧带，这是从附睾仔细分离。近提睾肌的顶点是小动脉和静脉，将它连接到附睾。阻断钳之间的这些船只，他们除了拉最大限度地减少出血。睾丸，附睾，睾丸动脉和静脉结扎近端（4-0或5-0丝线缝合），切断，连同相关的腹股沟脂肪垫丢弃（睾丸）。此外，睾丸可以轻轻推回腹腔，并用一块棉或Kimwipe保留。睾丸供应推入腹腔时，睾丸组织，以避免可能出现的创伤。我们没有发现明显的差异，在提睾肌微循环的准备的质量（见3.8节），无论是使用过程7,15。 </li><li>提睾肌的外表面被清除剩余的结缔组织，然后基座表面呈放射状蔓延。边缘固定在5-6地方创建一个完整的微循环横纹肌平板引脚（参见1.4节）。 </li><li>完成准备转移到一个活体显微镜的阶段，连续灌流（3-5毫升/分钟，34℃）和30分钟的平衡。 </li><li>提睾编制的完整性，是根据以下几个标准进行评估。自发以最小的手术创伤舒缩音是由动脉和静脉壁白细胞缺乏亮眼流表示。一个负责任的准备，最敏感的指标是提高动脉的灌流液5-10分钟的氧含量收缩。这是平衡与O 2的 21％（与5％O 2的 ;见第1.5节; 5％的CO 2，平衡N 2）。在动脉的白细胞存在，红血细胞通过毛细血管流动的情况下，和/或积累静脉及周围组织的白细胞组织损伤和炎症的迹象。 </li></ol> 4。提睾肌微循环的活体成像<ol><li>活体成像后，奥林巴斯MVX10立体声变焦平台基于一个自定义显微镜。 MVX10镜身安装到MVX10混合的立场与透明（科勒）成像（卤素灯），并配备与外延照明（汞灯）荧光成像。使用EPI -照明，GCaMP2兴奋30分之472纳米三十五分之五百二纳米收集排放。获得的图像通过一个MV PLAPO 2XC镜头（数值孔径= 0.50;奥林巴斯）使用XR/Mega-10加剧的数码相机（斯坦福Photonics公司，SPI）通过派控制软件（SPI），在个人电脑上。有了这个系统，观察视角（FOV）领域可以是多种多样的，从553微米X 442微米至22毫米x 18毫米。 </li><li>活体成像实验完成后，麻醉鼠标安乐死与颈椎脱位过量苯巴比妥。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/2874/2874fig1.jpg" alt="图1" /> 图1。鼠板鼠标提睾准备活体成像。一）与铝的平台楔形车身（黄色塑料绝缘），从底部看待。固定平台底部的加热电阻器进行热。二）加热平台的基础上的塑料楔。三）身体楔放在一个有机玻璃板手术，随后转移到的活体显微镜阶段。 Sylgard基座是用红色箭头表示。一个防水硅珠包围整个准备包含任何解决方案，在活体的过程中可能会泄漏，防止它滴在显微镜上。 <img src="/files/ftp_upload/2874/2874fig2.jpg" alt="图2" /> 图2:自定义宽视场成像MacroZoom显微镜。一）MVX10显微镜体XR/Mega-10 ICCD相机（奥林巴斯）（斯坦福大学光电子），安装在三目端口。 b）关闭视图显微镜体。 （一）变焦控制（0.63〜6.3X）;（二）滤光轮（三）图像倍频25X光学放大（NA = 0.50）。三）明（科勒）照明（冷凝器NA = 0.55，工作距离= 27毫米）的显微镜台下聚光器。 <img src="/files/ftp_upload/2874/2874fig3.jpg" alt="图3" /> 图3。完成鼠标提睾准备。在热烈的塑料涂层的铝平台（黄色）仰卧位麻醉鼠标。用胶带固定身体位置。提睾肌是呈放射状蔓延的透明硅橡胶的橡胶底座边缘的寄托。灌流的解决方案是在近端介绍，通过一个塑料滴头（白色箭头）。真空线（白箭头），删除解决方案通过Kimwipe灯芯。两个微量显示他们的秘诀在组织定位。参比电极（银线）是安全的提睾肌的下缘。 <img src="/files/ftp_upload/2874/2874fig4.jpg" alt="图4" /> 图4。龙虎斗动脉内皮细胞中的表达GCaMP2的网络可视化的放大倍率范围。一）荧光灯和B）明形象的3.2倍的光学放大倍率为总放大倍率= 42X在视频监视器上。 [视场（FOV）= 4375 x 3470微米]。比例尺= 500微米。在此放大倍率工作，有利于微量放置在所需位置。三）荧光和D）明影像总放大倍率6.4X光学放大倍率= 83X（视场= 2200 × 1759微米）。比例尺= 200微米。 E）荧光灯和F）明影像总放大倍率12.6X光学倍率= 165X（FOV = 1100 × 885微米）。比例尺= 100微米。 g）与图像倍频，光学倍率= 25.2X。比例尺= 50微米。

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+blood+flow+in+the+microcirculation:+Role+of+conducted+vasodilation.">Regulation of blood flow in the microcirculation: Role of conducted vasodilation.</a> Acta Physiol. , (2011).</li><li>Hill, M. A., Simpson, B. E., Meininger, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cremaster+muscle+hemodynamics+due+to+disruption+of+the+deferential+feed+vessels.">Altered cremaster muscle hemodynamics due to disruption of the deferential feed vessels.</a> Microvasc. Res. 39, 349-363 (1990).</li></ol>

所有的程序和协议，涉及动物，美国密苏里大学动物护理和使用委员会的批准，符合美国国立卫生研究院实验动物的照顾和使用指南进行。 制作这个视频文章已经由斯坦福大学光电子发起。

在这里，我们描述了在体内的微循环观察鼠标打开提睾肌准备。此过程是仿照"打开"睾准备首次在大鼠4。在不到1个小时就可以完成整个手术过程与实践。作为该制剂的多功能性使各种实验操作，很容易适应仓鼠和大鼠，实现了各种实验模型，要以类似的方式研究。准备工作是有限的雄性动物和测量应作出对中心的组织，避免损坏附近的肌肉 14边缘地区。它也应该认识到，虽然压力和流量分布改变时，提睾肌的互连船只被削减 16，微血管保持响应和适合再生的数据收集。可视化的提睾肌微循环的主要限制因素是结缔组织的建设，特别是在老鼠，但在仓鼠作为动物的成熟和大小增加。此外，由于动物发胖，它变得更加困难，因为苯巴比妥是亲脂性，可在脂肪组织中吸收，以控制麻醉。最好的办法是通过耐心，同时确保动物°的体温保持在〜37 ° C，而暴露组织与PSS的不断灌溉。

<table border="1"><tbody><tr><th>试剂或设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>氯化钠</td><td>费舍尔</td><td> S642 - 212 </td><td></td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P9541 </td><td></td></tr><tr><td>硫酸镁</td><td>西格玛</td><td> M2643 </td><td></td></tr><tr><td>氯化钙</td><td>西格玛</td><td> C1016 </td><td></td></tr><tr><td>碳酸氢钠</td><td>费舍尔</td><td> S233 </td><td></td></tr><tr><td>戊巴比妥钠溶液</td><td> Lundbeck公司</td><td> NDC 67386-501-55 </td><td>也简称为戊巴比妥钠</td></tr><tr><td>温度控制器</td><td>华纳仪器</td><td> TC - 344B </td><td>备用:可调节的12V直流电源</td></tr><tr><td> 20系列平台加热器套件RH - 2 </td><td>华纳仪器</td><td> 64-0274 </td><td>需要定制的铝板</td></tr><tr><td>兆丰- 10相机</td><td>斯坦福大学的光子学</td><td> XR/Mega-10 </td><td></td></tr><tr><td> MVX10 </td><td>奥林巴斯</td><td></td><td></td></tr><tr><td> MV PLAPO 2XC镜头</td><td>奥林巴斯</td><td></td><td></td></tr><tr><td> MVX10混合立场</td><td>利兹</td><td> LBX的混合</td><td></td></tr><tr><td>派控制软件</td><td>斯坦福大学的光子学</td><td></td><td>方案10万摄像头</td></tr><tr><td>立体显微镜</td><td>尼康</td><td> SMZ645 </td><td></td></tr><tr><td>防水硅酮密封胶（透明） </td><td>通用电气公司</td><td> 47970 - 72643 - LW​​5000 </td><td>其他明确的硅酮密封胶也工作</td></tr><tr><td> 60 × 15mm的培养皿</td><td>费舍尔</td><td> 08 - 757 - 13A </td><td></td></tr><tr><td> Sylgard </td><td>道康宁</td><td> 184 </td><td></td></tr><tr><td>显微切割剪刀</td><td>精细科学的工具</td><td> 15003-08 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>杜蒙钳</td><td>精细科学的工具</td><td> ＃5 / 45 </td><td></td></tr><tr><td> GP Millipore公司快递PLUS膜</td><td> Millipore公司</td><td> SCGPT05RE </td><td></td></tr><tr><td> Minutiens昆虫引脚</td><td>奥斯特利茨</td><td> M尺寸的0.15毫米</td><td></td></tr><tr><td>小型宠物微调</td><td>华尔推剪公司</td><td>型号9966 </td><td>每次使用后清洁</td></tr></tbody></table>

the mouse cremaster muscle preparation for intravital imaging of the microcirculation

整个身体，保持动态平衡，需要去除代谢副产品氧气和营养物质随之而来的不断的供应。实现这种平衡是通过血液微循环的运动，其中包括在整个所有组织和器官的血管供应的最小的分支。从动脉的动脉分支，形成网络，控制流入毛细血管的实质细胞密切相关的多种含氧血液的分布和幅度。毛细管提供了组织细胞的血液供应之间的扩散交换的表面积大。静脉收集毛细管污水和收敛，因为他们返回无氧对心脏的血液。实时观察这些过程，需要一个可视化和操纵的生活微循环的实验方法。 首次使用的组织学和电子显微镜后验尸1,2研究炎症模型大鼠提睾肌。第一次在体内的暴露完整的大鼠提睾肌报告调查反射光的使用血管活性药物的微血管反应。但曲率的肌肉和重点照明的缺乏限制了该制剂的实用性。重大突破，需要开放的肌肉，脱离睾丸和蔓延，为平板透径向下一个复合式显微镜4。虽然证明是一个宝贵的准备，以研究大鼠和仓鼠 6微循环的生理，提睾肌在小鼠7已被证明特别有用解剖参与调节8-11微血管功能和实时成像间的细胞通路信号12。 提睾肌是来自内部的倾斜和横向abdominus肌肉，睾丸下降通过腹股沟管13。它提供支持（希腊语:睾=吊杆）和维持睾丸的温度。此处所述，提睾肌准备作为一个优秀的光学分辨率平板薄。用鼠标保持在一个稳定的体温和麻醉平面，手术准备摆脱周围组织和睾丸的肌肉，蔓延到透明的硅橡胶橡胶底座，与昆虫引脚的边缘，而灌溉与生理盐溶液不断。本协议利用转基因小鼠动脉内皮细胞中表达GCaMP2。 GCaMP2是一个基因编码的荧光钙指标分子 12 。宽视场成像和加强电荷耦合器件摄像机， 使体内的钙信号在动脉内皮细胞的研究。

Watch this Scientific Journal Video about The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation at JoVE.com

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

一个组织准备用于可视化和生活微循环的实验操作。在麻醉雄性小鼠，薄，高度血管提睾肌准备活体显微镜来研究，包括动脉，毛细血管和静脉的微血管网络。本制剂对大鼠和仓鼠很容易适应。

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

the-mouse-cremaster-muscle-preparation-for-intravital-imaging

Research

JoVE Journal

Biology

32.2K 次观看.  University of Missouri. 一个组织准备用于可视化和生活微循环的实验操作。在麻醉雄性小鼠，薄，高度血管提睾肌准备活体显微镜来研究，包括动脉，毛细血管和静脉的微血管网络。本制剂对大鼠和仓鼠很容易适应。

视频: The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

科研

生物学

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis和显微操作的微循环活体荧光成像

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...