This work was supported by NIH Grant HL-113905

这项研究进行的所有动物的程序是由诺瓦东南大学的机构动物护理和使用委员会与指南实验动物的护理和使用一致认可， 第 8版（国家科学院出版社，华盛顿特区2011 ）。 1.准备组织切片受体放射自显影<ol><li>牺牲后，收获新鲜的脑组织，并尽快包装在铝箔并将其放置在-20℃的冰箱。要保持正确的形状，放置在大脑脑模具模拟头骨的内部，包裹在铝箔和发生在 - 20℃的冰箱中。 30分钟后，在可密封的冷冻储存袋处的组织和移动到-80℃冷冻长期储存。 <ol><li>获得的从-80℃冷冻，并转移到设定为最小的-10℃和最高的-18℃的低温恒温器，以避免解冻感兴趣的新鲜冰冻组织标本。 </li&gt; <li>放置在试样上组织与二醇和基于树脂的包埋介质装入，仅嵌入试样到培养基中的一小部分。对于大脑，大脑垂直安装，以使在冠状面切片。 </li></ol></li><li>将组织安装到低温恒温器内的切片机，并在地方拧紧。确保大脑的左侧和右侧是相同的前 - postero坐标，并且背腹轴垂直于常用脑图谱。 </li><li>开始在所需的厚度切割（20微米推荐）和解冻安装部分到显微镜载玻片在垂直方向为具有在滑动空间更大的表面积。收集部分在载玻片上以顺序每组五，即第一组幻灯片标记1-1,1-2，1-3,1-4和1-5（ 图3）。 </li><li>填写一组幻灯片与段后，允许幻灯片空气干燥达1小时，然后将SLI德在一个自密封冷冻储存袋在-20℃的塑料载玻片盒，和存储。 </li></ol> 2.放射自显影受体注意:放射性。使用防护服装来处理放射性。处置成立后依赖，并且必须遵循准则正确衰减（半衰期60天）或通过认证的公司被拾起。 <ol><li>从-20℃冷冻机中取出感兴趣的"-1"和"-2"幻灯片和安装到滑动夹（ 图5）。安装"-1"的幻灯片共同为"非特定的"治疗和"-2"幻灯片"总"的待遇。 "非特异性"罐子将包含10μM的PD123319的终浓度的AT 2 R拮抗剂氯沙坦的AT 1 R拮抗物，在测定培养基缓冲液（AM5）（ 表1）中 ，"总"结合罐子将只包含10 μMPD123319在AM5。 </li><li>反转幻灯片夹具放置载玻片到装有35-40毫升AM5的预培养科普林缸，和各自的抑制剂在室温下30分钟。在4分钟的时间间隔开始在他们的预培养浴随后集。 </li><li>立即在室温从预温育溶液的孵育滑动邮寄者传送幻灯片，含有10ml AM5的，再加上125 I SI-血管紧张素II的预定浓度与相应的抑制剂（ 图4中的计算），60-90分钟温度。如果有足够的放射性，10幻灯片可以放置（背靠背）在修改幻灯片夹和科普林罐子125 I-SI-血管紧张素Ⅱ培养。 </li><li>地方滑动回幻灯片夹具（如果需要），印迹，并冲洗轻轻1-2秒在400毫升蒸馏水在两个单独的容器中旋流。 </li><li>顺序地转移滑入含35-40毫升AM5的每个（如在图4 &lt;所示恰好1分钟4科普林罐子/ STRONG&gt;）。 </li><li>最后1分钟后用清水洗净，轻轻摇动部分在冰冷的蒸馏水中的四个转变1-2秒。 </li><li>吹干冷空气的幻灯片（注意:热空气会挥发放射性化合物）利用4分钟（ 图5）不同的角度设置了四个吹风机，直到所有的路段都干，然后将纸巾上。 </li><li>滑山与组织朝上到纸板的并置，以X光片使用双面胶带。 </li><li>安装的至少一个，125碘校准标准滑动到每个纸板（ 图6）。 注意:校准标准包括脑糊用125碘彻底混合绑定到含有苯酚环，通过离心在1ml结核菌素注射器压实的化合物，其是低温恒温器在相同的厚度脑切片切片并解冻安装到显微镜的幻灯片。另外，125我校准标准在20微米厚切片塑性树脂可以商购获得。在这些标准的塑料树脂部分地屏蔽来自辐射的膜，使得〜40％的组织等效应计入校准。 </li></ol> 3.胶片曝光与发展<ol><li>继续用皮带背透视卡带和放射自显影胶片暗房。打开纸盒，并把里面滑梯（ 图6）的硬纸板。 <ol><li>把灯关了，打开安全灯。小心打开X射线胶片的箱子，取出一部电影，并将电影光面向上（与右下角的锯齿边缘）在磁带上的幻灯片上。小心关闭磁带，并旋转锁定杆密封发光（ 图6）。在-20℃下暴露数日的幻灯片到几周。 </li></ol></li><li>在暗室中，打开磁带和胶片显影PROC进行ESS。 <ol><li>放置滑入连续托盘;显影剂2分钟，含有5％冰醋酸，30秒，和定影剂进​​行5分钟的水。该薄膜的置于与20分钟流水的托盘，然后放入Photoflo为不超过10秒，挂起。 注意:曝光时间凭经验确定，并且可以包括与不同曝光时间的多个片:长到足以从具有低结合区获得可测量的信号，但没有限制，只要通过与高结合区域饱和膜。一旦获得可接受的曝光，然后进行到下一个步骤。 </li></ol></li></ol> 4.组织学<ol><li>制备硫素染色和染色试剂（ 表2， 图7）。 <ol><li>放置"-3"在滑动架，和转让顺序用离子交换水开始1分钟，然后接着是三个倾角成去电离10分钟硫素染色液美化版水域，并在去离子一水30秒洗。 </li><li>继水，将滑动架成乙醇洗涤如下; 50％，70％，90％，30秒，然后在100％含双乙醇洗涤每个1分钟。最后，将滑动架成二甲苯为3分钟，然后转移到第二个二甲苯溶液5分钟。 </li></ol></li><li>在从最后二甲苯浴一次删除一个滑动，并覆盖与在有机溶剂中的安装平台的树脂基滑动件的上边缘，并放置一个24mm×60毫米盖玻片的幻灯片。允许幻灯片充分干燥〜48小时，然后扫描到电脑2400 dpi灰阶。 </li></ol> 5.光密度图像分析<ol><li>放置膜光泽面下来，用在左下角的锯齿边和扫描利用能够没有在成像系统的计算机的任何失真发送膜密度信息的专有扫描器。 </li><li>打开专用的成像SYS统和利用校准棒以建立校准标准（ 图9 - 12）在胶片上的图像的未来光密度分析。 </li><li>通过建立两种模板或经验概括感兴趣的领域的兴趣度的区域。数据被收集并基于膜分离，对照或野生型（部分），和区域。确保包括测量过程中的密度（飞摩尔/克），扫描区域和总目标区域（ 图9）。 <ol><li>通过确保感兴趣区域落在突出的参数（ 图10）之间调整扫描区域酒吧。 </li><li>导出数据到电子表格（ 表4）。乘以密度倍的总的目标区域，然后通过扫描区域，以获得用于结合该特定区域的值除以。一旦这样做是为了测量所有值，底物的非特异性的既定从总得到特异性结合p怨恨。平均值，也可以对这些值进行的。 </li></ol></li></ol>

Robert Speth has licensed antibodies to angiotensin II receptors to a commercial vendor and receives royalties from the sale of said antibodies. The other authors have nothing to disclose.

描述的协议标识"总"和"非特异性"在啮齿动物脑先前收获并保存在-80℃的冠状切片的相邻部分的放射性配体结合的可视化，并且可以容易地适用于几乎所有的组织中已解剖解决其中显示的受体或放射性配体结合位点的差的量子结构。在协议中描述的过程简单，分析是正确地解释结果的关键。测定20微米厚为最佳;如果部分过厚的非特异性结合会增加，从而难以解决的放射性配体的结合到它的目标。如...

心血管疾病仍然是死亡和残疾在美国，造成人员死亡，在美国超过30％的主要原因在2011年1，从美国心脏协会最近的统计数据表明，超过三分之一的人有一个或更多类型的心血管疾病。心血管研究继续迈大步对了解这种疾病，但作为一代开始变老当务之急是要继续努力。肾素-血管紧张素系统（RAS）的主要由促进动脉粥样硬化，炎症，全身性的血管收缩，和激活交感神经系统的（ 图1）2-8起着在心血管系统的调节中发挥中心作用。 在RAS是当肾的肾小球旁细胞分泌肾素进入血液响应于血压下降时启动的荷尔蒙系统，增加sympathetic刺激，或由致密斑流钠降低。肾素代谢血管紧张素原（在肝脏中合成），以形成血管紧张素I（血管紧张素Ⅰ）。血管紧张素I被随后通过血管紧张素转换酶（ACE）代谢，一种外酶对血管内皮细胞的腔侧，主要在肺，肾，以形成血管紧张素II（ANG II）中，在RAS的主要效应的肽。血管紧张素Ⅱ是能够激活两种接受亚型;的1型受体（AT 1 R）和2型受体（AT 2 R），两者调节心血管系统，保持体液和电解质平衡和现在被认为影响认知功能和神经变性疾病进程8,9。本地，特定脑-RAS报道独立合成血管紧张素II。在大脑中，前体蛋白血管紧张肽原在由肾素样酶3转化为血管紧张素我星形胶质细胞10合成，可能是肾素原结合于肾素原受体11，并且随后通过其在大脑中的神经元12的胞表面上大量表达血管紧张素转换酶转化成血管紧张素II。产生的血管紧张素II这intrabrain是对大脑的AT 1和AT个从血源性血管紧张素Ⅱ分离2受体的激动剂。 而对AT 1 R起着重要的生理作用，这是更好地了解整个身体的病理生理作用，主要影响心血管系统和肾脏（ 图2）。当血管紧张素II结合到AT 1 R，它会导致血管收缩;增加血流阻力和血压升高。它还促进醛固酮和加压素的合成和分泌，从而增加钠和水潴留。这些效果也可以引起缺血性脑损伤和认知障碍，并链接到帕金森氏病，阿尔茨海默氏病，和diabeTES，以及最近被鉴定为影响学习和记忆13-15。有在RAS在于一个反馈回路的AT在肾脏肾小球旁细胞1 R抑制肾素分泌。有趣的是，AT 2 R一般反调控的AT 1 R，导致在许多其他16-20血管扩张，轴突生长，轴突再生，抗增殖和cerebroprotection的动作。所述AT 2 R也被确定为抗高血压和最近，抗癌药21的靶。确定各种组织内的血管紧张素Ⅱ受体的定位和密度以及他们是如何通过定量光密度的受体放射自显影将有助于揭开RAS在特定疾病中扮演的角色不同的治疗方法和疾病状态的影响。 受体放射自显影已经使用了30多年作为一种有效的方法，用于指示血管紧张素I的存在下1受体和大脑中的RAS的其它部件和大鼠，小鼠，豚鼠，在各种实验条件下22-34狗和人的其他组织。大脑内定位血管紧张素II受体的重要性是一个可以应用功能的神经解剖学至血管紧张素II的动作在大脑中， 例如 ，AT 1 R的下丘脑（PVN）的室旁核的存在表明昂的函数II在大脑刺激加压素，催产素或促肾上腺皮质激素释放激素（CRH）释放，或交感神经系统的活化。因此，阻断对AT 1 R药物可能会降低一些与在脑的RAS活性相关这些PVN介导的作用。正在进行的工作表明，AT 1 R拮抗剂的使用可以减少创伤后应激障碍（PTSD）的CRH诱导释放和缓解创伤后应激障碍的症状（伤害等 ，提交出版）。在PVN，穹窿器官（SFO），和杏仁核被称为调节体内平衡，食欲/口渴，睡眠，记忆，情绪反应，并且是本实证研究的目标区域。通过收集在显微镜玻片上脑的冠状切片，并治疗与特异性抑制剂的部分与血管紧张素II受体具有特异性放射性配体沿检查这些区域。在这项研究中，与建议的厂商沿所有材料和试剂中列出，碘-125，采用放射性标记的血管紧张素II受体拮抗剂，肌氨酸1，异亮氨酸8血管紧张素II（SI血管紧张素II），然后将其作为单声道125我纯化-SI血管紧张素II使用如前面所述的35 HPLC方法。使用这种高比活性的放射性配体的允许的放射性标记的部分暴露于X射线胶片后低，中和高受体密度的区域的可视化。通过与含有已知量的碘-125，具体量的脑糊标准校准膜血管紧张素II受体的区域结合的进行量化。在实验研究中，血管紧张素Ⅱ受体实验对象的大脑结合可以比对照受试者的大脑。这可以指示是否血管紧张素II的动作响应于遗传条件，表型异常，疾病状态或药物治疗被改变。然后，该知识可以应用到疗法来治疗与RAS的失调相关的疾病的发展。标识受体结合位点的替代技术，但具有降低的解剖分辨率，被结合使用从组织匀浆，这是与放射性配体的范围内的浓度，以评估作为解离常数放射性配体结合亲和力的培养来源的组织膜制剂测定（ 杀敌 ）和最大结合能力（B 最大值 ）的感兴趣的组织的。 这里所描述的协议可以被分解成5大共mponents:准备组织切片受体放射自显影;受体放射自显影;胶片曝光和发展;组织学;和密度图像分析。

<table><tbody><tr><td>500 ml Plastic Beakers</td><td>Fisher</td><td>02-591-30</td><td></td></tr><tr><td>24 mm x 60 mm Coverslips</td><td>Fisher</td><td>22-050-25</td><td></td></tr><tr><td>Autoradiography Imaging Film 24 mm x 30 cm</td><td>Carestream-Biomax MR Film</td><td>891-2560</td><td></td></tr><tr><td>Bacitracin (from Bacillus licheniformis)</td><td>Sigma</td><td>B-0125</td><td></td></tr><tr><td>Cardboard Sheet 8 x 11</td><td>Crescent Illustration Board #201</td><td>201</td><td></td></tr><tr><td>Coplin Jars</td><td>Fisher Scientific</td><td>E94</td><td></td></tr><tr><td>Commercial hair dryers</td><td>Conair</td><td>Model SD6X</td><td></td></tr><tr><td>Disposable Culture Tubes</td><td>Fisher</td><td>14-961-26</td><td></td></tr><tr><td>EDTA (Disodium salt, Dihydrate)</td><td>USB Corporation</td><td>15-699</td><td></td></tr><tr><td>Ethanol</td><td>Fisher</td><td>16-100-210&amp;nbsp;</td><td></td></tr><tr><td>Formulary Substitute for D-19 Developer</td><td>Photographers Formulary, Inc.&amp;nbsp;</td><td>01-0036</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher</td><td>A38 SI-212</td><td></td></tr><tr><td>Histoprep/OCT</td><td>Fisher</td><td>SH75-125D</td><td></td></tr><tr><td>Film Fixer</td><td>Kodak</td><td>5160320</td><td></td></tr><tr><td>Photo flo</td><td>Kodak</td><td>1464502</td><td></td></tr><tr><td>Losartan</td><td>Fisher/Tocris</td><td>37-985-0</td><td></td></tr><tr><td>MCID&amp;trade; Core 7.0</td><td>MCID</td><td>N/A</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271</td><td></td></tr><tr><td>Peel-A-Way slide grips</td><td>VWR</td><td>48440-003</td><td></td></tr><tr><td>Permount</td><td>Fisher</td><td>SP15-100</td><td></td></tr><tr><td>PD123319</td><td>Fisher</td><td>13-615-0</td><td></td></tr><tr><td>Premium Charged Slides, Fine Ground Edge</td><td>Premiere Microscope Slides</td><td>9308W</td><td></td></tr><tr><td>&lt;sup&gt;125&lt;/sup&gt;I Ligands</td><td>Perkin Elmer</td><td>NEX- 248</td><td></td></tr><tr><td>&lt;sup&gt;125&lt;/sup&gt;SI-Ang II&amp;nbsp;</td><td>George Washington University</td><td>Radioiodinated by Dr. Speth</td><td></td></tr><tr><td>Slide Mailers</td><td>Fisher Scientific</td><td>HS15986</td><td></td></tr><tr><td>Sodium Dibasic Phosphate Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;PO4)</td><td>Fisher</td><td>RDCS0750500</td><td></td></tr><tr><td>Sodium Acetate (Anhydrous)</td><td>Fisher</td><td>BP333-500</td><td></td></tr><tr><td>Thionin&amp;nbsp;</td><td>Fisher</td><td>T409-25</td><td></td></tr><tr><td>X-Ray Casette (10 x 12)</td><td>Spectronics Corporation</td><td>Four Square</td><td></td></tr><tr><td>Xylene</td><td>Fisher&amp;nbsp;</td><td>X3P-1GAL</td><td></td></tr></tbody></table>

receptor autoradiography protocol for the localized visualization of angiotensin ii receptors

这个协议描述了使用特异于血管紧张素II受体进行受体放射自显影映射放射性为血管紧张素Ⅱ（血管紧张素II）中的大鼠脑受体结合模式。 组织标本收获并保存在-80℃。低温恒温器是用来冠部分组织（脑）和部分解冻 - 安装到带电的幻灯片。制作在载玻片上的组织切片在125 I SI-血管紧张素II孵育放射性标记血管紧张素Ⅱ受体。相邻的幻灯片被分成两组:"非特异性结合"非放射性标记的血管紧张素II的受体饱和的浓度，或存在（NSP）的AT 1血管紧张素II受体亚型（AT 1R）选择性血管紧张素II受体拮抗剂，与"总结合&#39;没有AT 1 R拮抗剂。 AT 2血管紧张素Ⅱ受体亚型（AT 2 R）饱和浓度的拮抗剂（PD123319，10μM）也出现在incubati关于缓冲区限制125 I-SI-血管紧张素II结合到AT 1 R亚型。在30分钟预温育在约22℃，NSP载玻片暴露于10μM的PD123319和氯沙坦，而&#39;总结合&#39;载玻片暴露于10μM的PD123319。载玻片然后用125 I SI-血管紧张素II在缓冲液中培养在PD123319存在关于&#39;总结合&#39;和PD123319和氯沙坦为NSP在测定缓冲液，随后几个"洗涤"，和水以除去盐和非特异性结合放射性。幻灯片使用吹塑机，然后暴露于放射自显影胶片使用一个专门的电影和录像带干燥。胶片显影和图像被扫描成用于使用专有成像系统和一个电子表格视觉和定量密度的计算机。另外一组幻灯片的硫堇染色进行组织学的比较。 使用受体放射自显影的优点是可视化的能力血管紧张素II受体原位 ，一个组织试样的一个区段内，和解剖学通过比较相邻的组织学参考部分确定的组织的区域。

所述肾素-血管紧张素系统的代谢途径的概要示于图1和直接集中在血管紧张素II受体亚型（AT 1 R和AT 2 R）在图2中进行说明。 图3显示冠状脑转移节到显微镜载玻片，然后将其通过一个受体放射自显影方法，使用预定的125 I SIANG II浓度运行， 如图4所示。 图5示出了用于在受体放射自显影?...

Watch this Scientific Journal Video about Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors at JoVE.com

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

receptor-autoradiography-protocol-for-localized-visualization

Research

JoVE Journal

Neuroscience

17.8K 次观看.  Nova Southeastern University. Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II. 

视频: Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书“

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

科研

神经科学

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

自动化研究动物交流的交互式视频播放

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

生物化学

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

免疫与感染

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

生物学

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of &#945;v&#946;3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

68加标记精氨酸甘氨酸 (rgd)-肽的血管生成

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

医学

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

使用 D11 scFv-碱性磷酸酶融合蛋白和免疫荧光直接检测组织中的异左旋蛋白

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to ...