这项工作是由Labex MemoLife并通过ARSEP支持（倒基金会欧莱雅助手点菜RECHERCHE河畔拉sclérose恩斑）

1.解决方案和材料<ol><li>准备隔离器的解决方案:B1，加1.5毫升HEPES为1M至150ml的HBSS; B2，加3.6克葡聚糖到20ml B1的; B3，加1克的BSA至100毫升B1的。 </li><li>通过切割掉底的上旋拧部分修改过滤器保持器。 </li><li>制备免疫染色方案:固定溶液，在pH 7.4的PBS的4％多聚甲醛;透/封闭溶液，稀释山羊血清至5％和Triton X100至0.25％的pH 7.4的PBS。 注意:固定取决于所用第一抗体的类型。 </li></ol> 2.解剖注意:无菌条件不是必需的，除非船舶用于细胞培养的目的。 <ol><li>准备150毫升的烧杯用20毫升B1的。置于冰上，并用封口膜覆盖，避免空气污染。 </li><li>深深麻醉鼠标引擎盖下的小纸巾浸泡在1毫升纯异氟醚被加入Int澳笼。麻醉是由于缺乏反应到足尖捏验证。杀死小鼠颈椎脱位。 注意:这些步骤都完成符合国家和机构的规定。 </li><li>可选:用20毫升的PBS 1X中进行心内灌注，消除血液中的含量24。 </li><li>第皮肤从颈部到鼻子手术刀将其拉离。删除所有污染的毛发，用PBS 1X。 </li><li>要打开颅骨，先插入剪刀前方的嗅球，并打开剪刀破裂的头骨两部分。 </li><li>小心取出用脑铲大脑。解剖出脉络丛从 ​​侧脑室，因为它们会污染血管制剂38。 注:可选:最后的准备工作将包括实质和脑膜血管。如果不希望的，脑膜可剥离以下由38描述的过程。 </li><li> TRansfer脑入含冰B1液的烧杯中。多达8个的大脑可以一起处理。 </li></ol> 3.脑组织均质<ol><li>使用两个解剖刀，在B1的溶液随后获得小块的约2mm手动大力击败大脑。 </li><li>均质化制剂用automatized Dounce匀浆，执行20笔在400rpm。确保玻璃管被保持在冰中，该douncer的上部是在溶液中移动时上下，从而防止气穴的形成。如果几个样品制备，每间同质化离子水冲洗douncer。 </li></ol> 4.血管净化<ol><li>转移匀浆到50ml的塑料管，然后继续离心以2,000rpm离心10分钟，在4℃。一块巨大的白色接口（主要是髓鞘）将形成对血管颗粒（红色的顶部，如果未进行灌注）。 </li><li>弃去上清液。该船颗粒和白色的接口将保持连接在一起。加入20毫升冰冷的B2解决方案，手动和大力摇晃管1分钟。 </li><li>进入第二离心4400克15分钟，在4℃。髓鞘现在将形成致密的白色层在上清液的表面上。 </li><li>仔细保持所述管和缓慢转动它，以允许上清液通过沿着壁分离从管壁髓鞘层。丢弃髓鞘与上清。保持附着在管的底部含有血管沉淀。 </li><li>吸干管的内壁与吸收纸缠有5毫升的塑料吸管和除去所有的残余液体，避免触及容器沉淀。保持在一个吸收纸管倒置排出任何剩余的液体。 </li><li>移液器向上和向下低结合的技巧，keepi暂停1毫升冰冷的B3解决方案的颗粒纳克管在冰上，再加入另外的5毫升B3的解决方案。确保船只分散尽可能并且不形成聚集体。 </li></ol> 5.过滤<ol><li>准备在冰上的烧杯用30ml冰冷却的溶液B3。用封口膜覆盖，避免空气污染。 </li><li>放置在修改后的过滤器保持器20微米筛网过滤器上的贝克尔烧瓶的顶部并通过施加将10ml冰冷B3溶液平衡。 </li><li>倒在过滤器上的船只准备和冲洗容器用40毫升冰冷的B3的解决方案。 </li><li>用干净的镊子恢复的过滤器，并立即沉浸在包含B3溶液的烧杯中。通过轻轻晃动松脱，从过滤器的船只。 </li><li>倒在50ml塑料管中并离心烧杯内容在2000克5分钟，在4℃。 </li><li>注意:可替换地，大脑血管&#39;从步骤4.6的悬浮液可以过滤到一个100微米的网过滤器。在这种情况下，较大的血管优先保留在过滤器上，而流过的包含微血管（主要是毛细血管），其然后在20微米的网过滤器过​​滤如上。 </li><li>悬浮微血管的沉淀在1ml冰冷却的溶液B3和吹打它至1.5 ml Eppendorf管中传送它。离心机在2000克5分钟，在4℃。 </li></ol> 6.固定，透和阻塞<ol><li>转船吹打成0.2毫升含有PBS使用1.0毫升低结合尖端PCR管。注意不要触及容器，因为它们可能会附着在笔尖的外部分。执行双目显微镜下的所有执行下列步骤。 </li><li>对于以下每个介质改变，离开管在冰上直到船只已达到的底部，并吸取出来最采用长和东西凝胶加载枪头的液体。 </li><li>除去大部分的PBS，并添加200微升固定液。暂停船只通过轻轻摇动孵育20分钟，在室温。 </li><li>吸取出固定液，并将其用200μl的1×PBS（第一次洗涤）的替换，孵育5分钟，在室温，重复此步骤3次。每次，验证血管已正确沉到管和移液管出来的1×PBS的底部，确保船只未触及。 </li><li>在最后一次洗涤后，更换1×PBS中由透/封闭溶液孵育1小时，在室温，通过轻轻摇动不时重悬血管。 </li></ol> 7.免疫染色<ol><li>更换透/阻断原发性抗体（在这里看到的和稀释液用在材料模板表的抗体的引用）稀释，在通透/封闭液的混合溶液中，孵育4°CO / N。 </li><li>洗涤3次后在PBS中在RT下，孵育第二抗体的混合物在PBS中稀释2小时的船只在RT。 </li><li>注:强烈建议进行核染色，用Hoechst（1:2000）:为了区分可能在显微镜下污染的毛或灰尘船（2000一）或DAPI。 </li><li>洗涤3次后在PBS中，重悬船只于50μlPBS中。 </li></ol> 8.安装和观测<ol><li>准备一个尖端硅化玻璃毛细管:用手术刀切P200的枪头和内部调整的毛细血管。毛细管的近似体积为40微升。 </li><li>使用硅化玻璃毛细管转移血管载玻片并小心地用一块吸收纸除去液体。 </li><li>应用安装中的单滴到血管和保持一个盖玻片在45°使降沿滑的边缘扩散。让熄灭盖玻片，并允许媒体传播缓慢。待其干燥O / N在室温避光。船只可以在一个fluoresc观察耳鼻喉科共聚焦显微镜。 </li></ol>

The authors have nothing to disclose.

血 - 脑屏障调节生理物质在中枢神经系统中的进出的通道和保护其不受存在于血液中潜在的有害物质。它参与几个CNS病症，包括神经退行性疾病2和脑肿瘤28。血脑屏障的渗透率极低的也阻碍了靶向神经细胞和方法意欲可逆打开血脑屏障无有害的后果对大脑的研究3一个非常活跃的研究领域的发展的治疗剂的通过。已作出许多努力来开发有价值模型和技术允许细胞，分子和药理学血脑屏障的调查，并且特别地，协议用于从啮齿动物的脑血管中分离。首先尝试简单地收集使用可变孔径大小的网格，从机械分离脑组织微血管丢弃污染脑实质细胞13，29，30，31。密度梯度离心以后被引入上尼龙网或在玻璃珠的列32，33，15相关的脑血管的过滤。在这些程序中，玻璃珠与尼龙网过滤似乎提高产量和高相对低速离心，微血管纯度，尽管高速离心还诱导剪切应力从 ​​而影响基因表达的15，32。酶消化的额外步骤也被介绍在一些协议在与分离粘附的神经元和星形胶质细胞以增加微血管纯度35的目的的解离步骤34。然而，过大的预消化也可能会破坏微毛细管完整性36,37。最近，免疫激光捕获显微切割（免疫LCM）已经显示出，以使即使在vesse不同的细胞群的船只或检索LS，检查改变血脑屏障的基因表达，尽管材料通过这种技术获得的量是非常有限的23。 在这里，我们提出了一个程序，脑血管的机械隔离由优素福等 [14]初步阐述。它提供获得纯脑血管，并根据其直径将它们分开的可能性。该协议不会勉强让整个大脑血管腔隙的净化，而我们并没有其疗效与之前公布的方案进行比较。然而，由于没有梯度，特定列和高速离心步骤使整个过程非常容易和便宜。重要的是，我们强烈建议删除脉络丛的大脑同质化之前所描述的38，因为我们经历过的污染的可能来源。其他重要的建议是:1）利用"低结合"塑料脑膜IAL（特别是枪头），因为船只坚持自然，未经处理的塑料。忽略这一点会导致大部分船只的损失; 2）小心平衡之前过滤尼龙滤膜（步骤5.2）; 3）粒料在尽可能多的缓冲尽可能过滤前的再悬浮，以提高样品的纯度; 3）如果有必要，血细胞和截留在纯化的血管蛋白质可洗出通过心内灌注用PBS前脑解剖; 4）纯化容器的产率可能通过重复从髓鞘分离在葡聚糖两次或三次（步骤4.2）39,40得到改善。对于免疫染色，我们强烈建议:1），以污染毛发和灰尘为此抗体有很大的非特异性亲和区分容器进行核标签; 2）避免离心分离收集容器各媒体的变化，因为它可能会导致材料的不可分割的球才形成。 的完整的脑血管纯化呈现很大的优势，研究血脑屏障的分子特征。虽然具体的或富集的内皮细胞和周细胞基因和途径已确定个别细胞类型18,19转录和蛋白质组研究中，公知的是离解和细胞分选技术导致细胞极性和形态的损失以及之间的互连细胞类型，这强烈地影响其分子谱。在这种情况下，保持整个血管腔隙完好如这里所描述，除星形胶质细胞，可能代表一个很大的优势。此外，相对于细胞分选，脑血管的纯化不需要使用特定的转基因报告小鼠品系或冗长免疫染色为基础的程序。另一个优点是利用大脑血管的是适应纯化协议对其他物种如已经示出了murine14的可能性，bovine13以及在人类中41,42。最后，本技术的进一步发展将是体外培养的脑血管片段。纯化的血管与原代星形细胞神经元和小胶质细胞的共培养可能有助于重新组装神经血管单元在比复杂的三维的多细胞培养系统，其中细胞首先分离和纯化被重组43.因此纯化的完整的脑血管之前一个准确的方式可能是任何类型的体外测定来研究血脑屏障的发展有很大的帮助。

中枢神经系统（CNS）的正常功能需要一个高度调节细胞外的环境中，以及相对于其他器官1其代谢需求是巨大的。中枢神经系统也是范围广泛的化学品，通常无害的外周器官，但它，神经毒性非常敏感。为确保正常运行，大部分的中枢神经系统"血管形成的血管内皮屏障;血-脑屏障（BBB），其控制分子和离子的流动，以及血液和大脑之间的免疫细胞的通路，从而保持适当的稳态2，也限制了治疗性药物的条目，从而妨碍治疗神经系统疾患3。在细胞水平，血脑屏障主要持续由内皮细胞，流出转运偏振光表达和非常低的胞转率4之间广泛紧密连接。性能及血脑屏障的功能大多是诱发NEighboring细胞4。具体地，周细胞发挥 ​​诱导和维持所述BBB 5,6中起重要作用。作为收缩细胞，周也调节血流量7做周围大血管的平滑肌细胞。最后，星形胶质细胞，脑的主要神经胶质细胞，发送名为endfeet围绕大型进程最大脑脉管8和调制血脑屏障完整性和免疫静止9，代谢物转移到神经元10，和诱导神经元活性之间的紧耦合和血流11,12。 研究脑血管系统的细胞和分子性质的能力是非常重要的特点更好其脑生理学和病理生理学的贡献。为了解决这个问题，已经发展战略，以隔离大脑的脑血管系统，该系统允许完整的脑血管片段的准备。脑血管purification被使用牛脑中13最初描述和改进，并适用于其它物种，特别是啮齿动物14。在这最后的研究中，使用不同大小的过滤器被引入到分离脑血管中，以级分富含不同直径的容器。有趣的是，在这些制剂，内皮细胞保持它们的代谢特性15，转运功能16和17的极化。在这里，我们详细描述了这个协议，并进一步证明了隔离容器保留大部分的原位结构。内皮细胞保持通过紧密连接相连，通过一个连续的基底层包围。周细胞和平滑肌细胞保持附着在内皮细胞层，以及血管周围星形胶质细胞膜。然而，星形胶质细胞，小神经胶质细胞，神经元和少突胶质细胞被消除。最后，我们描述了一个过程来执行免疫染色对离体脑血管。 </P&gt; 直至现在大多数关于脑血管系统的分子和细胞研究已经由解离纯化脑血管细胞进行细胞分选使用细胞特异性报道的小鼠品系或免疫染色基于程序18,19。尽管这些技术允许几乎纯脑血管的细胞群的分离，分离的细胞完全丧失其原位形态学和相互作用，这反过来又极大地影响它们的分子和细胞的特性。在这里描述的协议，从而允许整个脑血管片段无需特异性抗体或转基因小鼠的污渍的隔离，提供了一个很好的选择，作为分离的脑血管的总体结构是保守的，因此，减少它们的分子性能影响。隔离容器然后可以用于研究基因的活性，蛋白质合成和调节在所述BBB作为最近描述20,21 </suP&gt;。最后，相对于激光捕获显微切割22,23，本协议是廉价的，易于实施，并在任何实验室迅速适应。

<table border="0">
	<tbody>
		<tr>
			<td>Tissue Grinder Size C</td>
			<td>Thomas scientific</td>
			<td>3431E25</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge 5415&#160;R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td>5811000320</td>
			<td></td>
		</tr>
		<tr>
			<td>High-performance, Modular Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact System Provides High Quality Leica LED1000</td>
			<td>Leica</td>
			<td>LED1000</td>
			<td></td>
		</tr>
		<tr>
			<td>low binding tips (P1000)</td>
			<td>Sorenson BioScience</td>
			<td>14200T</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinnex 47mm filter holder PP 8/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=SX0004700">SX0004700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter disc Hydrophilic 20&#181;m 47mm 100/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=NY2004700">NY2004700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter disc Hydrophilic 100&#181;m 47mm 100/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=NY1H04700">NY1H04700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Wall Borosilicate Tubing</td>
			<td>Sutter Instrument</td>
			<td>B150-86-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>1014356290F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips, Thickness 1</td>
			<td>Thermo Scientific</td>
			<td>P10143263NR1</td>
			<td></td>
		</tr>
		<tr>
			<td>0,2 ml Thin-walled tubes and domed cap</td>
			<td>Thermo Scientific</td>
			<td>AB-0266</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;PARAFILM&#174; M (roll size 4 in. &#215; 125 ft)</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Life technology</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/14175129">14175-129</a></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life technology</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran from Leuconostoc spp. Mr ~70,000</td>
			<td>Sigma</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Euromedex</td>
			<td>ET330</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Formaldehyde (w/v), Methanol-free&#160;</td>
			<td>Thermo Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#8482; X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride (Hoechst)</td>
			<td>Sigma</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor&#174; 488 Conjugate; Dilution 1/100</td>
			<td>Life technology</td>
			<td>I21411</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrin (rabbit) ; dilution 1/400</td>
			<td colspan="2">kindly provided by Dr Markus A Ruegg</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti ZO-1 (mouse, clone 1A12)</td>
			<td>Life technology</td>
			<td>33-9100</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Smooth Muscle Actin (mouse, clone 1A4)</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti GFAP (mouse, clone GA5)</td>
			<td>Sigma</td>
			<td>G3893</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti AQP4 (rabbit)</td>
			<td>Sigma</td>
			<td>A5971</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Cx43 (mouse, Clone&#160; 2)</td>
			<td>BD Biosciences</td>
			<td>610061</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Olig2 (rabbit)</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>&#160;dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti NF-M (mouse)</td>
			<td colspan="2">provided by Dr Beat M. Riederer, University of Lausanne, Switzerland.</td>
			<td>&#160;dilution 1:10</td>
		</tr>
		<tr>
			<td>Anti Iba1 (rabbit)</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>&#160;dilution 1:400</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td>Life technology</td>
			<td>A11029</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate</td>
			<td>Life technology</td>
			<td>A11034</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 555 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td>Life technology</td>
			<td>A21424</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate</td>
			<td>Life technology</td>
			<td>A21429</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
	</tbody>
</table>

purification of mouse brain vessels

在大脑中，大部分的血管系统包括一个选择性屏障，血 - 脑屏障（BBB），调节脑和血液之间的分子和免疫细胞的交换。此外，庞大的神经细胞代谢的需求，需要一个时刻到时刻调节血流量。值得注意的是，这些规定的异常是最脑部病变的病因特点;包括成胶质细胞瘤，中风，水肿，癫痫症，退化性疾病（例如:帕金森氏病，阿尔茨海默氏病），脑肿瘤，以及炎症性疾病，如多发性硬化症，脑膜炎和脓毒症诱导的脑机能障碍。因此，了解的信号传导事件调制脑生理学是一个重大的挑战。多洞察构成该脑血管系统中的各种细胞类型的细胞和分子性质可以从原代培养物或细胞从新鲜分离的脑组织排序来获得。然而，如细胞极性，形态和细胞间的关系属性不保持在这样的准备。我们在这里描述的协议被设计成净化脑血管片段，同时保持结构的完整性。我们表明，隔离容器包括由是由一个连续的基底层包围紧密连接密封内皮细胞。周细胞，平滑肌细胞以及血管周围星形胶质细胞endfeet膜保持连接到内皮细胞层。最后，我们将介绍如何在纯化脑血管进行免疫染色实验。

在这里，我们描述了一种协议，允许为脑血管14的机械隔离。 图1概括了该技术的主要步骤。脑血管的结构是复杂的，并且包括几个细胞类型， 即，由紧密连接密封，并用周细胞，平滑肌细胞，和星形胶质细胞足突9包围内皮细胞。因此，下面的脑血管的隔离，我们的目的是通过如在我们的协议的第二部分中描述的免疫染色的纯化血管的结构表征。集聚蛋白，内皮基底膜的组分连续和均匀标记的在内皮表面（图2A）。透明occludens-1（ZO-1），内皮紧密连接的部件之一是免疫染色，没有明显的不连续，这表明内皮紧密连接被保存（图2B）。该周细胞内皮细胞覆盖，标签由NG2编，出现几乎连续如前所述25（图2C）。最后，平滑肌肌动蛋白标记显示致密平滑肌细胞层的存在下，在大的纯化的血管（图2D）的表面上。这些结果表明， 在原位脑血管结构的整体是保守在隔离罐片段。 星形胶质细胞已被证明发送大工艺，或&#39;endfeet&#39;到容器的表面上，护套几乎完全的脑血管系统 8。它们与容器紧密相互作用和其在维护和调节各种脑血管功能的作用，指定它们作为神经血管单元 9的一个重要组成部分。我们进一步分析孤立脑血管的星形胶质细胞血管覆盖。通常在整个星形胶质细胞免疫标记GFAP，本的星形细胞中间丝（图3A），华唯一的部分出现在显示一些短纤维的内皮细胞表面（ 图3B-C）纯化的船只。与此相反，连接蛋白43，高度富集血管周围星形胶质膜26间隙连接蛋白，在纯化的大血管（图3D）和毛细管（ 图3E）的表面的高度进行检测。同样地，水通道蛋白4（AQP4）的免疫标记，水通道家族的一个成员大大表示在面对容器27，26星形胶质细胞膜的水平，概述隔离容器（ 图3F-G）的壁。虽然星形胶质细胞没有共同纯化血管，脑血管分离的机械过程中保持附有他们的血管周围endfeet膜。 最后，我们通过其他的神经细胞类型检查我们的准备可能的污染。神经元中间丝免疫标记（NFM）（Hornun克等人，1999）揭示了附着在容器仅存纤维存在，这表明一个可能的共纯化的一些神经元端子（图4A-B）。小胶质细胞，标记的Iba1（图4C-D）和少突胶质细胞，标记的OLIG2（图4E-F），在隔离容器没有检测到。因此，神经元，小胶质细胞和少突胶质细胞没有共同纯化，用脑血管。 总之，这些结果表明，所描述的协议允许在结构上保留脑血管片段，在其上的血管周围星形细胞的膜保持附着的纯化。 <img alt="图1" src="/files/ftp_upload/53208/53208fig1.jpg" /> 脑血管净化议定书图1.摘要计划的主要步骤介绍。这个草图表示可以得到三种可能的容器馏分取决于音响<html>使用滤池（20或100微米目）。 S1:微血管和大血管，S2:大血管和S3:微血管<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53208/53208fig2.jpg" /> 图2.隔离容器保留大部分的原位结构。免疫组化染色孤立的小鼠脑血管的共聚焦图像投影。（A）基底层immunolabelled的集聚蛋白（红色）。（二）内皮细胞紧密连接immunolabelled的紧密连接（ZO-1 ） （红）。 （C）的周细胞immunolabelled为NG2（红色）。（D）的 immunolabelled对平滑肌肌动蛋白（SMA）的平滑肌细胞（红色）。细胞核用赫斯特（蓝色）。PG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53208/53208fig3.jpg" /> 图3.星形胶质细胞Endfeet膜保持连接到纯化的船舶。共聚焦图像投影。（A）大脑部分呈现出皮质血管免疫染色内皮细胞粘附分子（白色），星形胶质纤维酸性蛋白（红色）和连接蛋白43（Cx43的）（绿色的图像）（B，C）的免疫标记GFAP（绿色）在分离的毛细管标记的凝集素B4（白色的表面）（B标示平滑肌肌动蛋白（SMA）（红色）（C）的一个大容器中的）或。 （C）是一个孤立的毛细管标记的凝集素B4（白）（D）的表面重新打印的权限从20（D，E）Cx43的免疫标记（绿色） 或标示平滑肌肌动蛋白（SMA）的大容器（红色） <stroNG&gt;（E），（F，G），水通道蛋白4（AQP4）免疫标记（绿色），在一个孤立的毛细管标记的凝集素B4（白）（F）的表面或标示平滑肌肌动蛋白（SMA）的大型船只（红色）（G）。细胞核用赫斯特（蓝色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53208/53208fig4.jpg" /> 图4:神经元，小胶质细胞和少突胶质细胞的不共法用脑血管共聚焦图像投影（A，B）神经丝（NFM）免疫组化在20微米厚的海马脑片（红）（A）和纯化脑血管（ B），（C，B）在20微米厚的皮质切片小胶质Iba1免疫染色（RED）（C）和纯化脑血管（D），（E，F）在20微米厚的皮质片（红少突胶质细胞OLIG2免疫）（E）和纯化脑血管（F）核是由赫斯特染色（白），血管内皮细胞由Isolectine B4（IB4）（绿色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Purification of Mouse Brain Vessels at JoVE.com

Purification of Mouse Brain Vessels

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

23.3K 次观看.  Institut National de la Santé et de la Recherche Médicale. We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes. 

视频: Purification of Mouse Brain Vessels

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

与腺相关病毒的向量的生产、纯化和质量控制

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

富德瑞迪蒂菲洛波迪亚成分的纯化

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

从产后小鼠小脑中纯化Promin-1+干细胞

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

急性小鼠脑切片研究自发海马网络活动

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...