我们想确认为自动，电动四极管绞机设备在视频描述他的设计和建设，军山本。此外，我们要感谢所有过去和现在的威尔逊实验室成员的贡献微驱动器和四极管在实验室的技术开发。

1。制备四极管<ol><li>首先获得一个50厘米长的一个四极建设，聚酰亚胺绝缘，镍铬丝与一个直径为12.5微米的核心。折了一半的线。沿对你的手指，使它们粘在一起。确保对电线在其整个长度有良好的接触。 </li><li>对折，再由两端。确保一端形成的循环是不扭结。切使四个秘诀是一致的四个线对非循环结束。 </li><li>对于下一步，您将需要修改的鳄鱼夹，电动翻转装置，单杠和一个以上的转动设备。鳄鱼夹修改粘合塑料酒吧剪辑基地。凡接触线提示，钳四线，连同修改后的鳄鱼夹。挂在单杠的电线回路。将鳄鱼夹到机动阶段。 </li><li>应用80顺时针40计数器的时钟明智的曲折钢丝捆扎在约三分钟的过程中遵循的曲折。这些参数可以根据自己的需要是多种多样的。 </li><li>四极扭完成后，保险丝的电线加热热风枪（420 ° C或790 ° F）从三个不同角度，用中低流量。对于每一个角度，开始1-2厘米以下的电线束分裂，运行热枪放下，电线约5秒钟，以2厘米的距离。使用谨慎，因为高温会完全融化的绝缘，导致短路。 </li><li>现在的电线都融合在一起，取出轻轻提起鳄鱼夹，以纾缓紧张的四极的四极管，从捻线设备，并切断四极附近的鳄鱼夹。在另一端，切断回路，有四个非保税股导线长度相等。 </li><li>接下来，用软尖镊子轻轻弯曲的电线分开顶部的个人股。 </li><li>四极管是现在准备装载到微型驱动器阵列。 21日至25日四极管和存储在无尘框，直到它的四极加载过程的时间。 </li></ol> 2。装入四极管和接地线的微型驱动器阵列<ol><li>若要继续，您将需要一个完整的微型驱动器阵列。如果你还没有建，是指“慢性体内录音的微型驱动器阵列：驱动器制造”的视频在我们的设计方案，四极管会附着在一端连接器的硬件，将贯穿于聚酰亚胺在微驱动器等，电极提示以下的微型驱动器阵列的基础延长运营商管。 </li><li>在开始之前，建立一个驱动器支架上，一端可以连接到连接器板，并在另一端，可以通过一个panavise夹住。在这个例子中，驱动器支架上粘一个X - ACTO刀柄（图1c）的一端磨最大的连接器。 </li><li>与软尖镊子的四极，并使用一个立体，推入四极尖，聚酰亚胺载体管的微型驱动器之一。推入管的四极，直到个人的电极导线在驱动器阵列的顶部靠近连接器板。小心不扭结或弯曲的线，因为这将导致四极管在垂直角度，而不是进入大脑，并会削弱四极管的完整性。 </li><li>在电极板界面，使用软尖镊子轻轻送入各自的孔的四根导线的另一端​​。再次，避免扭曲或弯曲的电线。四线全部到位，推到他们的一双钳子，缩短下颌孔黄金引脚。当推入孔引脚，他们将剥离导线绝缘层，并创建一个电气连接。 </li><li>以供日后参考，微型驱动器和针电极上的接口板的位置之间的映射的轨道。 </li><li>继续加载共有18个四极管。其余三个微阵列上的驱动器将用于房子的参比电极。每个参比电极是由一个四极管，只使用一个四无粘结线两端。要附加的参比电极，遵循的加载过程，但只有四个电极中的一个附加到相应的参考电极针连接器上的板。 </li><li>如果您计划多现场录音，它可能是值得的，计划从四极管连接器板提前的连接。这应尽量减少在实验过程中调整了错误的四极的可能性。 </li><li>四极装载完成后，两个接地线，一个动物保护锥之一。切一个6英尺长的绝缘钢丝（文字：直径0.005“），并删除3毫米的绝缘，使用金属镊子，从每个线端。再切4“长期绝缘钢丝（文字：直径0.005”）。删除的一端和另一端的1厘米3毫米的绝缘。 </li><li>路线上的微型驱动器阵列的方孔，通过对接口板6“地线。在指定的地洞里的黄金引脚，裸露的电线连接电极接口板。此线将连接到该动物的头骨。 </li><li>路线4&#39;以前安装的接地线接地线平行。连接电极接口板，使用在另一个指定的地洞黄金针剥离3毫米年底。这根线，稍后将连接到一个保护锥，这将作为一个小的法拉第笼法，以减少噪声拾取。我们现在已经完成了连接线，可以继续完善我们的四极管。 </li></ol> 3。设置四极管的长度<ol><li>在这个过程中，我们的目标是切断所有的四极管，使他们略长的目标大脑结构的深度。在我们的例子中，设计的微型驱动器的最大行程为5-6毫米，足以达成许多新皮层区和成年大鼠背海马。首先，降低所有最大限度地暴露，使四极管微驱动器。同时切断所有四极管比期望的最终使用了一双锋利的罚款剪刀的长度较长，约5毫米的长度。 </li><li>晋级决赛，在panavise准备一双锯齿剪刀。夹一个很小的角度下，锯齿朝上一个句柄，而留下的其他处理晃来晃去。 </li><li>现在已经完全撤回到他们的引导套管转动的微驱动器的所有的四极管。 </li><li>使用一个立体，全面扩展了一个四极管及其套管。用一把尺子，位置锯齿剪刀，在指导导管所需的距离。慢慢地，轻轻地切断与一个平稳的运动四极。这四极管已被切断后，撤回的微型驱动器3 - 4mm的。每个四极重复这个过程，直到所有被切断。在这一点上，你准备镀金。 </li></ol> 4。镀金四极管和质量检查镀金的镍铬合金四极线是长期稳定录音的关键。它可以防止腐蚀和提高生物相容性。 <ol><li>安装在外部模式的阻抗米和直流电流发生器，音频输出（图1d）的检查模式。四极管内每个通道的六个可能对触摸的两根导线的电流发生器，使用音频反馈，以确定是否短裤对之间存在。如果存在短，重新再次减少锯齿剪刀和测试四极。如果问题仍然存在，丢弃的四极，取代它。 </li><li>当没有短裤存在于所有的四极管，将所有的微驱动器，尽量尽可能将所有的四极管。 </li><li>检查电极阻抗（图1e），修改设备配置。现在所有的四极管浸提示浴成一个标准的镀金解决方案。黄金浴电气连接阻抗计的积极的主导作用。检查通过触摸负阻抗计导致相应引脚连接器板电极的阻抗。 </li><li>测量和记录每个线对微驱动器阵列的阻抗。正常值范围从1-3兆欧。 </li><li>电流发生器，设置1-3之间μA的电流。下一步，牢固地附着在负极引线的阻抗计针连接器上的板。放入电镀配置的设备（图1F）。阻抗表上，迅速从正常模式切换，旁路模式，和背部。在旁路模式时，电流将通过四极管和黄金的解决方案，以及金将被镀上电极尖端。每一轮电镀后的阻抗应比以前少。重复的电流脉冲，如果阻抗不低于1兆欧。一个保守的范围内可接受的阻抗是250kOhm 350欧姆。如果低于200欧姆的阻抗下降，这可能表明短路。 </li><li>如果经过反复的脉冲的阻抗不降充分，可能会阻碍电极尖端。重剪的四极，短裤，检查和重复镀金的过程。如果不利于重切削，更换了四极管，并重新启动。 </li><li>电镀后的所有四极管，DIP简要地的四极提示，在乙醇中，并让他们干3〜5分钟。 </li><li>暴露在空气中的四极提示，四极管电流发生器内的短路测试。如果存在两个或两个以上的一个四极电极之间的直流连接，再削减黄金四极管，板。 </li><li>所有的四极管镀金完成后，添加一个中等厚度的氰基丙烯酸酯胶下降，每个tetrod之间的接口E及其聚酰亚胺管。这将确保它的微型驱动器的四极，并保持四极管的长度。 </li><li>作为最后一步，移动到驱动器所有的四极管，通过打开所有螺丝。 </li></ol> 5。附加的保护锥保护锥的目的是为了免受环境的微驱动器和暴露的四极线。它还提供了一个实验期间处理的支持，并减少电噪声。我们使用一个3 - D印刷塑料锥（参考第一部分：驱动器制造）。但是，它有可能使圆锥体像一个弯曲苏打片可以从其他材料。 <ol><li>以塑料锥，并修正了一些内部或外部表面铝箔胶带。 </li><li>接下来，通过锥钻一个小孔（直径1.5毫米）和松散地连接到一个螺丝钉，垫圈和螺母内锥。这将作为接地线的电器通过。我们建议使用0-80大小的螺钉和垫圈组合。 </li><li>首先，插入和部分变成圆锥体两侧的3个1-72大小的螺丝（长度在3 / 16英寸）。悬浮与驱动器阵列，在驱动器中插入锥。确保两个接地线从驱动器阵列的底部扩展。周围环绕的1-72螺丝几次之一4地线裸露部分，并紧紧地拧紧螺钉。圆锥体的安全驱动器基地，通过收紧其余两个锥形螺丝。 </li><li>最后，以1厘米剥离短的地线之间的铝箔胶带和垫圈，螺丝缭绕。拧紧螺丝固定地面。现在我们有一个最后准备手术植入的微型驱动器阵列。 </li></ol> 6。代表性的成果<ol><li>微驱动器阵列的生产，现已完成。你的微驱动器阵列装有18个四极管和3个参考通道。它是包裹在保护锥和耐久性第。 20克25g以内，平均完成的微驱动器阵列，带保护锥的重量。 </li><li>使用标准的外科手术植入完成的驱动器阵列。正如你可以看到，驱动器连接到前置放大器芯片和成捆的电线进行信号的数据采集硬件和软件。 </li></ol><img src="/files/ftp_upload/1098/CompositeFigure1_highqualityn.jpg" alt="图1" /> 图1：一个四极扭曲配置）示意图。一个单杠暂停上述自动化的四极扭装置的四极无粘结电极导线 B）四极阻抗测量电极电镀安装。检查短路时，电路不同的是：黑线连接的连接器板到另一个电极通道C）自定义一个近距离的微驱动器阵列持有人的工具。圆棒是一个X - ACTO刀柄。连接器是26针，双列，轧机最大的连接器。两件粘合在一起，使用牙科丙烯酸D）配置短检查。通道X和通道Y是四四极通道的任何两个不同的渠道。，五）测量电极阻抗的配置。电流源是“关闭”。阻抗计是“上”和“测试”。F）的黄金镀电极提示配置。电流源设置为&#39;on&#39;，音频为“ON”，模式是“单极”，直流测试为“ON”，目前设置为2微安。

<ol>
	<li>Gray, C. M., Maldonado, P. E., Wilson, M., McNaughton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodes+markedly+improve+the+reliability+and+yield+of+multiple+single-unit+isolation+from+multi-unit+recordings+in+cat+striate+cortex.">Tetrodes markedly improve the reliability and yield of multiple single-unit isolation from multi-unit recordings in cat striate cortex.</a> J. Neurosci. Methods. 63 (1-2), 43-54 (1995).</li></ol>

该协议是两部分协议的第二批：“微阵列的驱动器，用于慢性在体内录制。”相结合的微驱动器阵列和四极是获得一个清醒的许多神经元的同步录音功能强大的工具，表现动物准备。希望，我们已经提供了所有必要的信息，你会需要开始在自己的实验室或改进的电生理记录。 机动四极捻线设备定制的设计和制造。我们提供了一个供应商和部分的数量，将允许您要订购的具有?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Panavise</td>
<td>Tool</td>
<td>Panavise</td>
<td>Model 301</td>
<td>Holds either micro-drive array or serrated scissors</td>
</tr>
<tr>
<td>Modified Xcelite 378M Pliers</td>
<td>Tool</td>
<td>Newark</td>
<td>96F8903</td>
<td>For inserting connector board pins. To make, remove 2mm from one tip of the pliers.</td>
</tr>
<tr>
<td>Slow Cure Glue</td>
<td>Material</td>
<td>Great Planes (Champaign, IL)</td>
<td>GPMR6015</td>
<td>To glue the tetrode to the micro-drive</td>
</tr>
<tr>
<td>Gold Plating Solution</td>
<td>Material</td>
<td>Neuralynx</td>
<td>Sifco Selectron Gold NC</td>
<td>Contains gold particles for electroplating</td>
</tr>
<tr>
<td>Current Generator</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>A365D-B</td>
<td>A.K.A. Stimulus Isolator. Used to electroplate gold solution onto electrode tips.</td>
</tr>
<tr>
<td>Impedance Meter</td>
<td>Tool</td>
<td>BAK Electronics</td>
<td>IMP-1 / 6662 / 2788</td>
<td>To check impedance of electrodes</td>
</tr>
<tr>
<td>Hot Air Gun</td>
<td>Tool</td>
<td>Steinel (Germany)</td>
<td>HG 3002 LCD</td>
<td>To fuse electrodes together to make tetrode</td>
</tr>
<tr>
<td>Accu-Tek Carbofib Tip Tweezers</td>
<td>Tool</td>
<td>Aven (Ann Arbor, MI)</td>
<td>18768 (Pattern 304)</td>
<td>For handling tetrodes</td>
</tr>
<tr>
<td>Alligator Clip</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>18050-35</td>
<td>A.K.A. Bulldog serrefine. To clip the tetrodes together during twisting</td>
</tr>
<tr>
<td>Micro Dissecting Scissors</td>
<td>Tool</td>
<td>Biomedical Research Instruments</td>
<td>25-1000</td>
<td>For general cutting of tetrode/electrode wire</td>
</tr>
<tr>
<td>Iris Toughcut Serrated Scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14058-09</td>
<td>For final cut of tetrodes</td>
</tr>
<tr>
<td>Teflon Coated Stainless Steel Wire</td>
<td>Material</td>
<td>A-M Systems (Carlsborg, WA)</td>
<td>790500</td>
<td>Ground wire</td>
</tr>
<tr>
<td>12.5 micron nichrome wire</td>
<td>Material</td>
<td>Kanthal Palm Coast</td>
<td>Rediohm-800 1/4 HARD PAC COATING</td>
<td>Polyimide coated micro-wire for making tetrodes.</td>
</tr>
<tr>
<td>Tetrode Twister</td>
<td>Tool</td>
<td>Neuralynx</td>
<td>Tetrode Assembly Station</td>
<td>Increases the speed and consistency of tetrode fabrication.

</td>
</tr>
<tr>
<td>Silver Paint</td>
<td>Material</td>
<td>GC Electronics</td>
<td>22-023</td>
<td>For connecting the micro-electrode wires of the tetrode to the connector pins (see Discussion)</td>
</tr>
<tr>
<td>X-Acto Knife Handle</td>
<td>Material</td>
<td>MSC Direct</td>
<td>X3001</td>
<td>For constructing a micro-drive holding device.</td>
</tr>
<tr>
<td>26 Pin, 2 Row Connector</td>
<td>Material</td>
<td>Mill-max</td>
<td>852-10-026-30-001000</td>
<td>For constructing a micro-drive holding device.</td>
</tr>		</tbody>
		</table>
		</div>

micro-drive array for chronic in vivo recording: tetrode assembly

四极，捆绑四个电极，已被证明是一个有价值的工具，同时记录多个神经元在体内。差分幅度超过四极通道动作电位签名允许单机活动多单元信号的隔离。能够精确地控制脑立体定位的位置和四极管的深度，研究整个大脑区域的协调的神经活动的关键。在与一个微驱动器阵列相结合，这是可以实现精确定位和稳定的控制在几天或几周的过程中，许多四极管。在这个协议中，我们演示了如何制造和使用的基本工具和材料条件四极管，安装到一个长期的多驱动器的四极管阵列的四极管的录音，在大鼠，接地线连接的微型驱动器阵列，并在体内到的微驱动器阵列一个附加的保护为了保护环境与身体接触的四极管锥。

Watch this Scientific Journal Video about Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly at JoVE.com

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

在这个协议中，我们将演示如何制造和使用同一个微驱动器阵列，这是在大鼠慢性电生理记录设计条件四极管。此外，我们说明了微驱动器阵列的建设，其中包括安装接地线和保护锥的最后阶段。

慢性的微型驱动器阵列在体内录音：四极大会

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

micro-drive-array-for-chronic-in-vivo-recording-tetrode-assembly

Research

JoVE Journal

Biology

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

33.1K 次观看.  MIT - Massachusetts Institute of Technology. 在这个协议中，我们将演示如何制造和使用同一个微驱动器阵列，这是在大鼠慢性电生理记录设计条件四极管。此外，我们说明了微驱动器阵列的建设，其中包括安装接地线和保护锥的最后阶段。

视频: Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

科研

生物学

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

一种慢性脑功能成像开颅手术过程

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性的微型驱动器阵列在体内录音：驱动器的制造

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...