这项工作是由巴黎第六大学和皮埃尔·法布尔创新基金的支持。

所有的程序均按照照顾和使用实验动物的欧盟标准进行，与当地的伦理委员会进行动物实验（巴黎大区 - 巴黎委员会，授权4270）的批准。 1.麻醉<ol><li> （最多至1mg / 10g体重）腹膜内（27号针和1毫升注射器）到成年小鼠手术前注入戊巴比妥的致死剂量。 </li></ol> 2.血管灌注注:没有必要血管灌注过程中兽医药膏敷眼睛。这个过程是快速（5-10分钟），并在动物死亡结束。确认缺乏应对与脚趾捏的。 <ol><li>使用虹膜剪，使一个切口，约4厘米长，入腹壁和腹膜，只是肋条笼之下。 </li><li>做一个小切口（几毫米长）的膜片，然后continuË沿肋骨的整个长度隔膜的切口以暴露胸膜腔。 </li><li>提起胸骨路程，钳止血胸骨尖;把脖子上的止血钳。精心修剪的脂肪组织连接胸骨心脏。 </li><li>穿过左心室15号针头灌注到心脏的顶点。 </li><li>最后，用剪刀剪下肝叶之一来创建一个出口。 注:另一种插座可以通过使用虹膜剪创建到右心房切口创建。 </li><li>灌注动物用一个泵以2.5毫升/分钟的速率操作25至50ml的磷酸盐缓冲盐水（PBS）的。肝脏应作为灼血被替换为PBS。 </li><li>约五分钟后，一旦从肝脏中的流体是完全清楚，停止灌注。 </li><li>如果免疫染色或常规染色计划，灌注所述动物用50ml的多聚甲醛（PFA;在PBS中的4％），15分钟。 注意:小心，PFA烟雾是有毒的。用PFA动物灌注应在通风橱中进行。 </li></ol> 3.脑的分离和Willis环<ol><li> 脑的隔离 <ol><li>用一双手术剪取下头上。 </li><li>请与虹膜剪正中切口，沿颈部到鼻子皮肤。 </li><li>切去皮肤暴露的头骨和清除残留的肌肉和脂肪组织与虹膜剪。 </li><li>虹膜剪的锋利端放入枕大孔一侧，并沿头骨到外耳道（也称为耳道）的内表面上小心地将它们。 </li><li>重现对侧在3.1.4中描述的切口，使沿顶叶间骨的内表面切成矢状缝的开始的中线。 </li><li>工厂虹膜剪刀额骨，右两眼之间，在矢状缝，然后打开它们一分为二头骨。 </li><li>提起出大脑，抢夺嗅球，并使用虹膜剪刀剪下其腹面的神经连接。 </li><li>取出大脑，并放置在含有冰冷的PBS为牛隔离60毫米的培养皿。完全沉浸在PBS的大脑。如果大脑固定用4％PFA（用于随后的切片和免疫染色或定期染色），保持它在4％PFA的浴中于4℃24小时。 </li></ol></li><li> Willis环的分离 注:解剖显微镜需要奶牛隔离。大脑应该在整个过程保持在4℃。 <ol><li>把脑倒挂（ 即在其背水面），以可视化的奶牛。 </li><li>用小镊子在嗅叶的基部抢前脑动脉（ACA）（ <str翁&gt;一个，图1）和施加压力，以从容器中连续分离它们。使用相同的步骤，以切割在b中的大脑中动脉（MCA）（ 图1）。 </li><li>使用镊子锋利的两端提起并取下主要干道形成了从皮质牛。 </li><li>抬起后交通动脉（PCA）的开始从大脑断开它们，通过抓握大脑中动脉（MCA）与钳子。拿起前动脉（ACA和MCA），轻轻地拉他们在多前 - 背方向视交叉。为了防止牛中断，中断处理与其他血管的过程。 </li><li>重复步骤3.2.2和3.2.3上级和后小脑动脉（SCA）/（PCA）（ 三，图1）和用于基底动脉（BA）（D，图1），在一个dorsal-拉动它们前方向。停在过程中的描述我的结束ñ3.2.4。 </li><li>通过使用镊子轻轻拉动取下整个牛。将牛在60毫米培养皿中充满了冰冷的PBS并用两个镊子取出任何剩余的连接脑组织，拿着牛的地方小针。 </li><li>保持收获的母牛在-80℃下用于RNA纯化后续处​​理（RNA提取产量大量的RNA - 约500纳克）或蛋白质提取。 注意:牛可维持离体 24小时，通过调整对分离的小鼠嗅动脉开发的器官浴系统7。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54352/54352fig1.jpg" /> 图1:对小鼠脑突出的牛 ，牛是从两个内部颈动脉（MCA），这是从两个大脑前动脉（ACA）派生形成的腹面示意图。基底动脉（BA）分支成后（PCA）和优异的（SCA）的脑动脉和两个椎动脉（VA）。

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The authors have nothing to disclose.

我们在这里描述为Willis环的隔离可再现协议。涉及牛最常见的脑血管疾病是CAA相关血管病变，颅内动脉粥样硬化和颅内动脉瘤，所有这些都影响动脉血管的壁。的危险因素是众所周知的，但这些脑疾病的分子发病机制仍然知之甚少和用于预测它们发生特定的生物标记物所缺乏的。目前的方法相当大的兴趣了隔离转基因小鼠的牛的肉眼观察结果与分子变化联系起来。例如，通过在其特定的基因被敲除（如由保坂等人 8描述的）的小鼠模型诱导一致动脉瘤和分析牛，它应该是能确定治疗是否定位由该基因编码的蛋白质可以防止颅内动脉瘤和/或蛛网膜下腔出血。 Obviou狡猾，这种方法也可用于分析特定药物对CAA相关血管病变的AD的各种转基因小鼠模型的发展中的作用。 这是比较容易分离小鼠动脉比净化整个小鼠微血管，但这样的抽样仍然是在小鼠中比在大鼠更加困难。事实上，鼠牛嵌在硬得多脑膜，从而有可能在一气呵成整个结构隔离。除了在小鼠血管的小尺寸的问题，这个过程也棘手由于除去大脑前以下其用PBS灌注容器的透明度。因此，我们建议没有练习输液，麻醉后的第3步开始，使其更容易区分血管。最后，老鼠脑血管非常细小，容易折断，解剖应非常仔细地进行，没有冲过来，以保证整个结构被隔离在一块，首先抓住ACA。牛制剂的纯度可以通过比较神经和血管的基因的表达水平进行评估。通过练习，单头牛只提供5的研究足够的RNA - 10基因的表达。对于大型的筛选，几个鼠标母牛应合并。 高塞尔等人 9描述的方法用于分离小块微血管（基于使用胶原酶/分散酶的消化来捕获它们大脑玻璃珠和片段），该产生平滑肌或内皮细胞可在培养时保持放置在适当的培养基中。我们的方法的优点是，它分离大血管，而不改变血管结构。奶牛的特定分支的解剖也可能感兴趣，以确定一个特定分支的基因表达谱和可能的易感性脑diseas之间的相关性ES，如颅内动脉瘤。这些方法可以提供一个解释，为什么一些ACA的解剖和遗传变异与ACA动脉瘤的发病率较高有关。这种夹层要求的压力在每一特定分支的基地钳施加，从其他部门分离的，在牛分离过程的开始。 因此，除了使得有可能从奶牛10派生的细胞或用于筛选基因表达的11和蛋白质生产12或翻译后蛋白修饰，可以通过简单地调整为开发的器官浴系统中使用的用于离体研究中，这种方法离体小鼠嗅动脉。7按照在含有抗生素的培养基中的整个牛的温育后，可以获取并分析由该特定结构释放的分泌组。这种分泌蛋白质在CAA的小鼠模型的分析将使POssible，例如，以确定这些脑动脉的CAA相关炎症状态。所得条件培养基也可以被用来确定在每个血管壁的细胞类型的表型的分泌组的效果。病理牛制剂也可用于识别在生化信号的可能差异，如环鸟苷酸（cGMP），环磷酸腺苷（cAMP），或Ca 2+浓度下，用荧光共振能量转移（FRET）检测的基于生物传感器。病毒递送的生物传感器已被用于在含有实验获得成熟活的神经元与保存形态脑切片制剂，导致检测的在锥体皮层神经元和纹状体中型多棘神经元之间的D1响应深刻的差异。13 在转基因小鼠脑血管疾病的表达基于FRET的第二信使传感器蛋白14建模生化信号中分离出来的牛成像应进一步提供进一步洞察生化，病理生理学，和这些疾病的药物。

脑底动脉环（circulus动脉脑瘤），也被称为威利斯（COW），Willisor威利斯多边形的环圈）最早是由托马斯·威利斯在1664描述的是周围设视交叉和下丘脑，可以循环系统吻合被看作是一个中心毂供给血液到脑和周围结构。血经颈内动脉和椎动脉进入这个结构，它通过内部中，后脑动脉流出圈。每个这些动脉的具有左右分支圆上的任一侧。基底，后连通，并且前交通动脉完全的圆（ 图1和图2）。血流受损的任何流出动脉的风险由血液进入从颈和脑动脉的圆，从而保证有足够的血液供给到b中的合并减少雨。这种结构也可作为在颈内动脉的严重闭塞性疾病侧支血流的主要途径。 几种类型的脑血管疾病有他们的牛奶原产地。最常见的是脑淀粉样血管病（CAA）相关血管病变，颅内动脉粥样硬化和颅内动脉瘤。1，2，3，这些疾病可能导致灌注不足是由于血管扩张，和脑和/或蛛网膜下腔出血最终转化为缺血性或出血性中风或充其量短暂性缺血发作。在诊断过程，包括神经影像学，可能与血管造影术相结合的最新进展，已使人们有可能在临床诊断这些主要脑血管疾病，而不需要一个脑活检。然而，有效和特定的治疗（药理学或血管内），目前缺乏，因此，有必要定义新分子靶点。 的新颖的药物靶标用于预防这些疾病在人类中鉴定需要的动物模型并分离脑脉管系统包括牛的方法。这些模型应提供的证据和线索的具体变化，包括炎性改变，在颅内动脉瘤，CAA或颅内动脉粥样硬化的动物模型的大血管壁发生。4，5，6 我们已经建立了小鼠牛隔离的方法，以促进在阿尔茨海默氏病（AD）和相关疾病容器炎症研究，例如CAA。该隔离鼠标牛方法，为脑血管炎症基因表达的疾病进展过程中的评估开发。再加上检测软脑膜及脑膜动脉壁内的β淀粉样蛋白的沉积，这种方法可以更容易地阻止矿山在脑血管壁的炎症基因表达和Aβ肽的积累之间的可能关系。大脑，包括蛛网膜下腔的柔脑膜和软脑膜的血管网，是大动脉形成Willis环的扩展。此处所描述的方法可以用于任何小鼠谱系的牛隔离和可用于在大型船只的小鼠脑脉管系统的所有类型的筛选（ 例如 ，基因表达，蛋白质产生和翻译后蛋白修饰）的。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Hardened Fine Iris Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Scissors - Straight / Sharp / Sharp&amp;nbsp;&amp;nbsp; 16.5 cm</td><td>Fine Science Tools</td><td>14002-16</td><td></td></tr><tr><td>Dumont #7b Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11270-20</td><td></td></tr><tr><td>Stereoscopic Zoom Microscope</td><td>Nikon</td><td>SMZ745T</td><td></td></tr><tr><td>CellBIND Surface 60mm Culture Dish</td><td>Corning</td><td>#3295</td><td></td></tr><tr><td>Peristaltic Pump - MINIPULS 3</td><td>Gilson</td><td>M312</td><td></td></tr><tr><td>Pentobarbital Sodique</td><td>Ceva Sant&amp;eacute; Animale</td><td>FR/V/2770465 3/1992</td><td></td></tr></tbody></table>

protocol for isolating the mouse circle of willis

脑底动脉环（circulus动脉脑瘤）或威利斯（COW）的圆是围绕着视交叉和下丘脑的血液供应到大脑和周围结构的循环吻合。它已牵涉几种脑血管疾病，包括脑淀粉样血管病（CAA）相关血管病变，颅内动脉粥样硬化和颅内动脉瘤。这些疾病的基本为新颖的药物靶标对他们的预防识别的分子机制的研究需要的动物模型。其中一些模型可以是转基因，而其他将涉及脑血管的隔离，包括这里所描述的CoW.The方法适合于在任何小鼠谱系牛隔离，并具有相当大的潜力用于筛选（基因，蛋白质生产的表达，翻译后蛋白修饰，分泌组分析等 ）上的大血管鼠标cerebro-研究血管。它也可以用于体外研究，通过调整对分离的小鼠嗅动脉开发的器官浴系统。

<html>在PBS-灌注小鼠被杀害，并且在协议中的第3.2节所述的奶牛被隔离。当正确地进行清扫，奶牛应该出来在一块和应稍透明由于在脉管没有残留血液。 <img alt="图2" src="/files/ftp_upload/54352/54352fig2.jpg" /> 图2: 隔离后的母牛鼠标。在一个10厘米的培养皿中的牛（一）概述（二）奶牛的各个分支的详细信息。 MCA大脑中动脉，ACA为大脑前动脉，BA为基底动脉，PCA为大脑后动脉和SCA优越的脑动脉。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54352/54352fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 的帐篷。"FO:保持-together.within页="1"&gt;牛制剂的纯度可以通过确保特定血管基因被强烈表达，而神经元的基因的表达是检测不到的核对更具体地，清洗牛应特异性表达的血管平滑肌细胞标记，如平滑肌肌动蛋白（SMA），平滑肌肌球蛋白重链（SM-MHC）和smoothelin。这些标记在脑的其它部分几乎没有表达。表达的相反图案应神经元基因获得，与神经元标记（MOG，MAP2）只在牛几乎检测不到。 mRNA的水平可以通过RT-qPCR的与相对于参照基因转录物归一化（这里，即GAPDH的）来评估。典型的结果示于图3A和B。 <img alt="图3" src="/files/ftp_upload/54352/54352fig3.jpg" /&gt; 图 3: 在威利斯的鼠标圈神经和收缩基因和脑组织表达的RT-qPCR的结果归反对那些参考基因（GAPDH）。它们被表示为平均值±6至12个独立实验的SD;用脑牛的比较:NS，不显著，*，P &lt;0.05，** P &lt;0.005，和***，P &lt;0.001（A）收缩基因的表达:SMA（平滑肌肌动蛋白） smoothelin，SM-MHC（平滑肌肌球蛋白重链11）和E-selectin（B）中的神经元基因的表达:莫古（髓鞘少突胶质糖蛋白）和地图2（微管相关蛋白2）。 在缺乏牛脑内皮标记物E-选择的表达模式非常类似于对牛的样品获得，这可能反映了脑毛细血管网的存在。 <strong&gt;的表1列出了每个成绩单的平均值的Cp数字。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 一个。 </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2"> 平均的Cp </td><td colspan="3"> GAPDH </td><td colspan="3"> SMA </td><td colspan="3"> Smoothelin </td><td colspan="3"> SM-MHC </td><td colspan="3"> E-SEL </td></tr><tr><td> 意思 </td><td> SD </td><td> ñ </td><td> 意思 </td><td> SD </td><td> ñ </td><td> 意思 </td><td> SD </td><td> ñ </STRONG&gt; </td><td> 意思 </td><td> SD </td><td> ñ </td><td> 意思 </td><td> SD </td><td> ñ </td></tr><tr><td> 牛 </td><td> 20.86 </td><td> 0.93 </td><td> 12 </td><td> 22.02 </td><td> 0.67 </td><td> 12 </td><td> 25.29 </td><td> 1.89 </td><td> 12 </td><td> 23.37 </td><td> 0.80 </td><td> 12 </td><td> 30.56 </td><td> 1.66 </td><td> 12 </td></tr><tr><td> 脑 </td><td> 18.71 </td><td> 0.72 </td><td> 6 </td><td> 33.91 </td><td> 2.06 </td><td> 6 </td><td> 31.08 </td><td> 3.17 </td><td> 6 </td><td> 26.05 </td><td> 0.73 </td><td> 6 </td><td> 28.45 </td><td> 0.63 </td><td> 6 </td></tr><tr><td> B. </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td> &lt;TD&gt; </td><td></td><td></td><td></td></tr><tr><td rowspan="2"> 平均的Cp </td><td colspan="3"> GAPDH </td><td colspan="3"> 猫 </td><td colspan="3"> MAP2 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 意思 </td><td> SD </td><td> ñ </td><td> 意思 </td><td> SD </td><td> ñ </td><td> 意思 </td><td> SD </td><td> ñ </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 牛 </td><td> 21.04 </td><td> 0.41 </td><td> 6 </td><td> 32.13 </td><td> 1.63 </td><td> 6 </td><td> 30.21 </td><td> 0.85 </td><td> 6 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 脑 </td><td> 19.09 </td><td> 0.49 </td><td> 6 </td><td> 26.43 </td><td> 0.88 </td><td> 6 </td><td> 23.30 </td><td> 0.99 </td><td> 6 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表 1: 平均交叉点的神经元和收缩基因在威利斯的鼠标圈中的表达与脑的计算是基于交叉点（CP）的荧光水平不变确定精确的周期比较。交叉点是对应于所述扩增曲线的最大二阶导数的循环数。更高的Cp值与较低水平的表达相关联。对于给定的基因，如果CP超过参考基因的35（A）中的Cp表达被认为是不可检测:GAPDH;和收缩的基因:SMA，smoothelin，SM-MHC和E-选择（B）参照基因的CP:。GAPDH;和神经元基因:莫古和地图2。

Watch this Scientific Journal Video about Protocol for Isolating the Mouse Circle of Willis at JoVE.com

Protocol for Isolating the Mouse Circle of Willis

We describe here a reproducible protocol for isolating the mouse circle of Willis.

协议隔离威利斯的鼠标圈

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

protocol-for-isolating-the-mouse-circle-of-willis

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

13.0K 次观看.  Sorbonne Universités. We describe here a reproducible protocol for isolating the mouse circle of Willis.

视频: Protocol for Isolating the Mouse Circle of Willis

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

科研

医学

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

免疫与感染

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

小鼠的共性共性隔离和识别

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

离体下丘脑神经元的激素、代谢和电调节

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As the functions of the hypothalamus are controlled by specific subsets of neuronal populations, the ability to isolate them provides a major tool for studying metabolic mechanisms. In this regard, the neuronal complexity of the hypothalamus poses exceptional challenges. 
For these reasons, new techniques, such as Magnetic-Activated Cell Sorting (MACS), have been explored. This paper describes a new application of magnetic-activated cell sorting (MACS) using microbead technology to isolate a targeted neuronal population from prenatal mice brains. The technique is simple and guarantees a highly pure and viable primary hypothalamic neuron culture with high reproducibility. The hypothalamus is gently dissociated, neurons are selectively isolated and separated from glial cells, and finally, using a specific antibody for a cell surface marker, the population of interest is selected. 
Once isolated, targeted neurons can be used to investigate their morphological, electrical, and endocrine characteristics and their responses in normal or pathological conditions. Furthermore, given the variegated roles of the hypothalamus in regulating feeding, metabolism, stress, sleep, and motivation, a closer look at targeted and region-specific neurons may provide insight into their tasks in this complex environment.

作者聚焦：下丘脑神经机制洞察 

Here we present a protocol to grow specific hypothalamic cell subtypes in culture. The cells can be selected based on opportune/unique membrane markers and used in many applications, including immunofluorescence, electrophysiological, and biochemical assays.

分离靶向下丘脑神经元，用于激素、代谢和电调节研究

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As ...

神经科学

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 ...

生物学

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

面对小鼠血液制备

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

协议隔离威利斯的鼠标圈

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此视频中的章节

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

小鼠的共性共性隔离和识别

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

分离靶向下丘脑神经元，用于激素、代谢和电调节研究

分离靶向下丘脑神经元，用于激素、代谢和电调节研究

分离靶向下丘脑神经元，用于激素、代谢和电调节研究

鼠冠状动脉血管平滑肌细胞的分离

面对小鼠血液制备

一种持续分离小鼠心肌细胞的简单有效的方法