该项目是由NIH授予U01HL098180从支持国家心脏，肺和血液研究所。内容完全是作者的责任，并不一定代表官方意见，国家心脏，肺和血液研究所或国立卫生研究院

1。试剂设置 1.1解剖和成像介质 Leibovitz的L-15培养基（L15）补充有FBS（10％）和青霉素 - 链霉素（100单位/ ml青霉素G钠和100微克/毫升硫酸链霉素）气管样品收获和成像过程中使用的。 2。浅寨文化商会大会 图1所示的腔室用来装气管组织。室的地板是玻璃见底35毫米培养皿。通过为0.3mm厚的硅薄片的玻璃盘上方放置一小片产生的腔室的顶部和侧面。在片材的边缘切断，以适应的圆底盘和中心进一步修整，以形成一个浅的中央腔室（见步骤2.1-2.3）。除在此组件中，一个18毫米的圆形盖玻片下产生一个封闭的腔室，适合于成像。 <ol><li>完全覆盖了18毫米的圆形玻璃盖玻片0.3毫米厚的硅薄片的正方形。 </li><li>随着标准的锐性剥离剪刀剪掉多余的矽胶片，从盖玻片的边缘，（矽胶片层在一个圆形的）。 </li><li>随着锋利的手术刀切出来，取出矽胶片（约10平方毫米），从中间盖盖玻片（由此形成的顶部和两侧的成像室）中心广场。 </li></ol> 3。气管收获和准备<ol><li>安乐死小鼠根据本地机构动物保健和使用委员会和/或政府指引，并有足够的L15媒体，泅组织（作者只是在出生前在妊娠16.5天的小鼠，通过对去除气管成一个标准的塑料35毫米培养皿新生儿，新生儿和成人的动物2）。 </li><li>删除非气管内有相关的组织附着到外的气管使用钝性分离。 </li><li>取出喉与横向切割正下方的环状软骨使用微清扫剪刀（ 图2A中 ，第1行）。 </li><li>拆下的左，右主支气管分支与的横向切割正上方的隆突使用微清扫剪刀（ 图2A中 ，第2行）。 </li><li>轻轻握住气管用细镊子和冲洗管腔2-3次〜1毫升的沐浴L15介质与P1000和P1000的Pipetman尖清除粘液和血液。 </li><li>切出3-4环段气管采用横向切割，微解剖剪刀（ 图2B，1号线）。 </li><li>通过使用微解剖剪刀（ 图2B 2号线），这短短的气管段气管肌肉的中间纵向切开。 </li><li>通过纵向切开气管软骨环的中间使用微解剖剪刀（ 图2C </stro纳克&gt;，行），离开气管组织适合成像的两个部分（ 如图2D所示的单节）。 </li></ol> 4。纤毛成像<ol><li>传输气管组织流明段，面朝下，到中间的L-15培养基100-200微升35毫米的玻璃底培养皿。 </li><li>为了量化纤毛生成的流洗澡气管组织样品（〜1×10 11颗粒/ ml或20微升/毫升的Fluoresbrite 2.5％的微球悬浮液的水溶液）的L-15培养基中，添加少量荧光0.20微米的微球。 </li><li>将预先制备的（见上述第1至第3步骤）上方的气管样品硅胶片的一面朝下的摄像室，位于浅的壁形成的腔室由窗口矽胶片切成在中间气管样品（ 图1 ）。 </li><li>盖玻片上轻轻向下推以固定到位，并消除任何多余的L-15媒体从边缘全光照GA尖P1000和P1000的Pipetman。 </li><li>安装35毫米的玻璃底培养皿和气管样本100X客观和DIC的光学显微镜上。 </li><li>使用一个高速照相机和电影采集软件收集电影纤毛蠕动在200 +帧每秒沿气管纤毛上皮细胞的卷曲边缘气管肌肉（ 图2D概述箱，例如E：静止图像录制电影） （ 补充影片1）。 </li><li>使用FITC啶成像和低光CCD摄像头（见上面的设备清单），收集荧光微球运动的电影，整个气管纤毛上皮表面的纤毛产生的流量（ 电影2）允许后定量。 </li></ol> 5。定量纤毛运动<ol><li>负载DIC成像ImageJ软件程序包，由I所描述的技术的电影转换成泉德拉蒙德13使用直线工具绘制光栅线穿越跳动的纤毛。 A“”这条线重新划分产生kymograph的跨线的纤毛运动产生波浪纹型图像。每个波峰之间的像素数是他们测量（一个像素的=一部电影帧），然后可以计算每分钟节拍数（ 即赫兹）。 </li><li>为了测量纤毛产生的流量负荷荧光珠电影成的ImageJ软件包，并使用的MTrackJ插件14手动跟踪整个气管上皮细胞表面的荧光珠，并让软件生成钢丝速度和方向性跟踪的每个胎圈。 </li></ol>应小心，当使用荧光珠到量化流作为打浆纤毛的距离，可以强烈地影响测量流量的大小。珠补充运动电影2，这是一个很好的例子。在这个例子中的胎圈打浆纤毛的表面移动速度很快，并显着下降的有孔玻璃珠进一步相差在入浴媒体。为了控制这个问题，应该只收集珠描记在一个固定的距离，在所有样品中的纤毛上皮细胞，以允许正确地进行比较。最后，取得对流体流动的有代表性的数据，有孔玻璃珠应尽可能长的时间的跟踪，我们优选使用用于计算流体流动，覆盖10 +纤毛细胞的胎圈轨道。

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作者什么都没有透露。

测量纤毛摆动频率（CBF）是比较容易的使用高倍显微镜的目标和快速的图像采集硬件13,15，并解释了为什么脑血流量测量形成的基础研究在健康和疾病中的黏液纤毛清除。然而，当脑血流量测量是必不可少的了解黏液纤毛清除，单独测量CBF睫状肌产生的流量和纤毛摆动波形，这两者都是比较难以衡量，往往被忽视的重要性，忽略了潜在的，可能没有相关的脑血流量。例如如何增加脑血流量...

阐明遗传和环境因素会影响粘液纤毛清除功能和肺的健康1，气道上皮细胞的运动纤毛功能分析实验是重要的。成像的小鼠气道上皮细胞的简单的协议，开发提供了一种有效的方法来审问呼吸道纤毛运动的突变体和基因敲除小鼠模型，并在小鼠气管组织解剖和体外成像与高清晰度电视显微镜，呼吸道纤毛蠕动只需要基本技能。此协议在建立和完善了大规模的鼠标诱变屏幕，运动纤毛功能允许快速评估（纤毛摆动频率，纤毛摆动形状，纤毛产生的流量）患有先天性心脏疾病的伴有内脏异位2-5的突变体。 目前的技 ​​术，用于研究呼吸道纤毛蠕动，可以分组为急性体外类型或离子蒙古包长期在体外实验方法。急性实验包括人/鼻气道活检刷6,7 体外可视化和分析简单的横向气道第8。 在体外细胞培养方法利用各种技术，以产生分化的纤毛上皮细胞，如空气中的液体界面的文化或气悬浮培养9-11张。然而，这些气道上皮reciliation的技术要求非常显著的投资在时间和培训之前任何可用的纤毛上皮细胞的实验（4-6周9,10）。虽然通常用于人体临床研究急性呼吸道上皮刷活检体外分析，这种方法是不是可以使用小鼠的研究，由于加剧了机械组织损伤12。 在这个协议中所述的技术分析的谅解备忘录ê气管的气道上皮细胞不仅是简单的执行，但它不需要特殊的解剖技能，也没有任何专门的设备除了那些电视显微镜成像标准。这个简单的协议有许多优点。首先，小鼠气管组织收获是快速和容易执行，它允许大量的小鼠气道纤毛功能快速评估。这可以包括急性分析不同在体外治疗的短期效果。其次，作为体外技术，气道纤毛上皮细胞仍然连接到相关的支持组织和，从而保留相关的细胞信号通路。因此，在比较， 在体外 reciliated的气道上皮细胞，该制剂在体内的组织环境是一个更好的代表性的自然。第三，该协议允许收购了一些不同的量化参数，可以提供客观的评估运动纤毛f油膏。最后，在对比其他流动呼吸道纤毛可视化的方法，这个协议允许的纤毛纤毛摆动方向直角可视化，允许纵断面图中的纤毛，是最佳的高分辨率成像的纤毛摆动和metachronal的波代。 这个协议可以修改了一些方法来解决了广泛的实验需求等作用的药物制剂，遗传因素，环境暴露，和/或机械因素，如粘液负载上呼吸道纤毛功能和生成/维护呼吸道纤毛摆动和波传播metachronal。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium </td>
 <td>Invitrogen </td>
 <td>21083-027 </td>
 <td>No phenol red </td>
 </tr>
 <tr>
 <td>Fetal bovine serum </td>
 <td>Hyclone </td>
 <td>SH30088.03 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin </td>
 <td>Invitrogen </td>
 <td>15140-122 </td>
 <td></td>
 </tr>
 <tr>
 <td>2x fine forceps </td>
 <td>Roboz </td>
 <td>RS-4976 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5676 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5620 </td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel </td>
 <td>Roboz </td>
 <td>RS-9801-15 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 pipetman </td>
 <td>Gilson, Inc </td>
 <td>F123602 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 tips </td>
 <td>Molecular BioProducts </td>
 <td>2079E </td>
 <td></td>
 </tr>
 <tr>
 <td>18 mm round glass cover slips </td>
 <td>Fisher Scientific </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 35 mm culture dishes </td>
 <td>Corning </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Glass bottom 35 mm culture dishes </td>
 <td>Warner Instruments </td>
 <td>W3 64-0758 </td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone sheet 0.012" (0.3 mm) thick </td>
 <td>AAA Acme Rubber Co </td>
 <td>CASS-.012X36-63908 </td>
 <td></td>
 </tr>
 <tr>
 <td>0.20 &mu;m diameter Fluoresbrite YG Carboxylate Microspheres </td>
 <td>Polysciences </td>
 <td>09834-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope, with 100x oil objective and DIC filters </td>
 <td>Lecia</td>
 <td>DMIRE2 </td>
 <td>Brand is not critical. </td>
 </tr>
 <tr>
 <td>100-watt mercury lamp, epifluorescent FITC excitation/emission filters</td>
 <td>Lecia </td>
 <td></td>
 <td>Brand is not critical.</td>
 </tr>
 <tr>
 <td>Microscope stage Incubator</td>
 <td>Lecia </td>
 <td>11521749 </td>
 <td>Not required if imaging cilia at room temperature</td>
 </tr>
 <tr>
 <td>High-speed camera bright field </td>
 <td>Vision Research </td>
 <td>Phantom v4.2 </td>
 <td>Brand is not critical. Must be faster than 125 fps</td>
 </tr>
 <tr>
 <td>High-speed fluorescent camera </td>
 <td>Hamamatsu </td>
 <td>C9100-12 </td>
 <td>Brand is not critical. Must be faster than 10 fps</td>
 </tr>
 <tr>
 <td>Movie analysis software</td>
 <td>National Institutes of Health</td>
 <td>ImageJ with MtrackJ plugin</td>
 <td></td>
 </tr>
 </tbody>
</table>

ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia

体外小鼠气道上皮细胞成像技术定量分析洞察黏液纤毛清除功能的重要运动纤毛功能已经确立。刚收获的小鼠气管，通过纵向切开气管肌肉和安装在浅壁室玻璃底的菜。其长轴沿气管样品趁气管肌肉纵向卷曲。这使得成像的纤毛运动，沿着整个气管长度在纵断面图。使用微分干涉显微镜和一个高速的数码相机得到允许纤毛摆动频率的定量分析和睫状波形画在200帧/秒。在成像过程中添加的荧光珠，纤毛产生的流体流动也可以被确定。协议时间跨越约30分钟，5分钟5-10分钟的样品制备室安装，和10-15分钟电视显微镜。

控制呼吸道纤毛应清晰可见，看到击败以协调的方式（ 补充电影1，电影播放放缓至15％的实际时间），纤毛摆动的方向（ 补充电影2，电影播放100％有明显的流实时）。定量的纤毛电影应该产生类似的结果如图3所示。未能收集高速DIC电影使纤毛摆动频率的定量分析（ 图3C），纤毛摆动形状的定性评估，并给出了良好的图象，而荧光珠的跟踪允许?...

Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

一个简单而可靠的可视化和量化技术，气道纤毛的蠕动和纤毛产生流量使用小鼠气管。这种技术可以被修改，以确定如何广泛的因素，包括药物制剂，遗传因素，环境暴露，和/或机械因素，如粘液负载影响纤毛运动。

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

ex-vivo-method-for-high-resolution-imaging-cilia-motility-rodent

Research

JoVE Journal

Biology

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

20.7K 次观看.  University of Pittsburgh. 一个简单而可靠的可视化和量化技术，气道纤毛的蠕动和纤毛产生流量使用小鼠气管。这种技术可以被修改，以确定如何广泛的因素，包括药物制剂，遗传因素，环境暴露，和/或机械因素，如粘液负载影响纤毛运动。

视频: Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

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 ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...