作者要感谢来自NIH / NINDS R01 NS069861和NJCBIR CBIR14IRG024到VS.支持

1.电期间用biocytin灌装注:读者可以参阅基本膜片钳技术和仪器19-22，这是不经这里阐述替代来源。这里详述的步骤假定为膜片钳记录的设备和方法已经确立，并且描述将被限制到生物胞素填充和事后免疫相关的信息。在这个手稿列出所有实验对老鼠进行的。 <ol><li> 350微米23 -使用振动，在厚度为300制备所需区域的活脑切片。 </li><li>加入2毫克生物胞素，以1毫升的内部溶液6的制备含有0.2％生物胞素的内部溶液。为了达到最佳效果，超声处理10内部的解决方案 - 15分钟，直到生物胞素完全溶解。接着，装入一个1毫升的注射器装配有0.2的内部溶液＃160;微米孔径的聚丙烯过滤嘴附连到微加载。保持在一个寒冷的包这个负荷的注射器保持镁ATP和Na-GTP的内部解决方案的稳定性。 注意:一个含有内部溶液葡糖酸钾（126毫K - 葡糖酸盐，4毫米氯化钾，10mM的HEPES，4mM的镁ATP，0.3mM的钠GTP，和10mM磷酸）是最佳的某些神经化学标记物的回收，包括小清蛋白，免疫标记中。根据我们的经验，利用铯和氯为基础的解决方案，内部以降低免疫染色恢复某些神经化学标志物的能力。 Neurobiotin或细胞非透性，可固定，极性示踪剂一个Alexa荧光团与生物胞素结合可被用作替代生物胞素。 </li><li>在红外微分干涉对比，直观记录相应的单元格。为了尽量减少轴突任何中断，由沿使用"方法"F其轴线降低吸移管接近电池主体油膏在大多数显微可用。采用膜片钳生理协议建立19-22红外线下的微分干涉对比全细胞记录。 注:避免从推定轴突的方向接近的小区，因为它会切断轴突，形象化为在切割端（ 图1中的绿色箭头）一个泡。此外，避免施加高正压录制吸管，而接近小区，以减少生物胞素的溢出，这将导致高背景染色。 </li></ol><img alt="图1" src="/files/ftp_upload/54880/54880fig1.jpg" /> 图1.建议平面接近细胞的用biocytin填充。该图像示出了（使用594缀合的链霉红色）一颗粒细胞（GC）与已被处理以显示生物胞素记录期间生物胞素填充。由GC有它在XY平面内取向的树突。注意切断颗粒细胞轴突（绿色箭头）由于沿XY平面移液器运动。请注意，这是理想的沿XZ平面接近这个神经元。比例尺:50微米。 <ol start="4"><li>在电压钳结构中，确定全细胞电容和串联电阻响应于小（5毫伏），短暂（30毫秒）电压使用利用在浴模式的膜测试功能在一个电生理学数据采集和分析软件的步骤。这将确保建立全细胞记录条件，使生物胞素填充。 </li><li>根据需要进行在任一电流或电压钳模式生理记录。在34°C&gt; 10分钟的最小记录时间是理想的。 可能需要更长的记录时间，在室温下进行的研究:注意。 </li><li>在生理录音结束后，通过缓慢移动小步记录吸管，交替向上（沿Z轴）重新建立膜片钳配置和向外（沿X轴）在电压钳模式。 </li><li>同时监测使用"膜测试"功能来可视化电容瞬变的损失和直线的当前响应的塌陷的电容和输入电阻，表示该小区的重新密封，并建立一个外输出的修补在枪头。保持细胞在去极化电位（-40毫伏）将促进再次密封过程。 注:此过程为清除细胞的通常确保胞体和树突的完全恢复。在此过程中或者在分离从吸管的细胞不施加正压到记录吸管。在切片的细胞记录的胞体的可视化表示成功脱离。细胞碎片或在分离的尖的端部的膜的膜片的存在通常表明胞体的错位，不会产生完整细胞形态。 </li><li>前前后后器从细胞分离的吸移管中，保留在记录室中的切片为3 - 5分钟以确保染料的远端树突和轴突过程的传输。 </li><li>转移切片含有4％PFA中固定24孔板中。 注意:PFA是致癌性，和适当的个人防护设备，包括手套和口罩，必须使用，以避免刺激皮肤和吸入。 PFA易燃，必须保持火灾接触。 PFA是从未在排水布置的，并且必须被收集为化学废物。 PFA固定必须从活切片准备利用，避免生理设置的污染领域，包括移液管被孤立。 </li><li> 24 - PFA固定48小时后，对存储的切片转移至0.1M磷酸盐缓冲盐水（PBS）免疫染色之前。 注意:理想情况下，生物胞素恢复和免疫时最好固定后不久进行的，虽然每个染色<html>固定收益率的好成绩7天之内形成的。片可以在PBS维持0.02 - 0.05％的叠氮化钠固定后进行染色达到和超过90天。虽然在PFA过夜固定适用于大多数抗体，它是可能的一些抗体时培养在固定剂的持续时间被减少最好。固定在PFA超过48小时减少抗原的可用性，并减少对神经化学标记成功二次免疫的机会。 注意:叠氮化钠是极其危险的，在与皮肤或眼睛或摄入后或吸入接触刺激物。严重的过度曝光可能会导致死亡。该化合物的致癌性和致突变性是未知的。使用包括手套，防护眼镜，白大褂，和防尘口罩适当的个人防护装备。叠氮化钠是从未在排水布置的，并且必须被收集为化学废物。 </li></ol>e_title"&gt; 2。第一个主抗体和用biocytin染色 （1天） 注意:下面的免疫染色步骤为自由浮动的部分，并且需要连续以低速（2转/分钟，转速）对所有孵育步骤振荡器振荡。 <ol><li>洗组织3次，每次10分钟在0.1M PBS（pH值= 7.4）。 注:0.1M PBS中可以商业购买或取得在实验室如下（使1升）:8克NaCl的将0.2g的KCl，1.44克Na 2 HPO 4的，及0.24g的KH 2 PO 4在1大号卫生署2 0。 注意:如果所需的神经化学标记物是已知的，染色的蛋白/可同时使用生物胞素染色进行肽（步骤2.2 - 2.4）。如果染色只生物胞素，继续执行步骤2.5。 </li><li>可选:阻止10％封闭血清（BS;在RT在0.3％的Triton X-100稀释于PBS 1小时，正常山羊血清（NGS））。 注:每口井应该接收溶液的相同体积推荐量是250之间 - 500微升/孔。阻断血清的选择是基于在该第二抗体被升高（ 例如，NGS用于染料缀合的山羊抗- （各自的主抗体种））的物种。如有需要使用在不同的物种中产生免疫染色切片二级抗体，包括在阻断步骤（2）在每个正常血清的10％，每个正常血清用于初级和二级抗体孵育步骤的3％。 </li><li> （可选）孵育一抗的章节中，CB 1 R（多克隆，豚鼠）在0.1M的PBS 0.3％的Triton X-100，和3％BS在室温O / N。 CB 1 R被用来识别大麻素受体1型表达。 注:此步骤是可选的，不需要揭示生物胞素馅。 图2示出在第一染色过程标记为生物胞素和CB 1 R的部分。 O / N INCUbation在RT足以满足大多数的初级抗体;然而，某些初级抗体，包括CB 1R，需要3 -温育5天。如果孵育需要长于1天，则建议在4℃下温育，因为的Triton X-100可透片，在室温下延长温育过程中可能会导致组织的瓦解。包括缺乏一级抗体在至少一个部分，用于每个系列的实验，作为二次抗体的特异性的控制的阴性对照。为阴性对照，初级抗体被省略，但在0.3％的Triton X-100的0.1M PBS中和BS被添加。 </li></ol><img alt="图2" src="/files/ftp_upload/54880/54880fig2.jpg" /> 图2. 成功CCK染色1周之后用biocytin染色和C的恢复 annabinoid受体1型（CB 1 R） - &lt;STRONG&gt;标签。 （A - C）在60X共聚焦图像显示了生物胞填充神经元（A）和CB 1 R免疫反应（B），由箭头指示。同样的部分为1周（C）后CCK免疫染色处理。图像叠加显示在D中。 CCK免疫反应使用远红透露和青色为伪彩色。 （ 五 ）A.注意生物胞填充细胞的形态重建的生物胞素和CB 1 R染色甚至第二CCK免疫染色后是显而易见的。还注意到CB 1 R和CCK免疫染色模式的预期分布。 （F -高 ）放大从细胞轴突的形象，如在A，生物胞素的节目紧密合作，定位和轴突CB 1 R（箭头）。比例尺:50微米（A - D），100微米（E）和10微米（ <strong&gt;的F - H）。图片由Yu 等转载。 2015年9。 <a href="http://ecsource.jove.com/files/ftp_upload/54880/54880fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> （第2天） <ol start="4"><li>洗脑切片3倍，每次10分钟在0.1M PBS（pH值= 7.4）。 </li><li>孵育在链霉红染料缀合物的部分（浓度:1:1000;激发:与在可见红光光谱发射594纳米）与在0.1M PBS中0.3％的Triton X-100和3％BSA在4℃的CO / N的暗（用铝箔包裹）以显示生物胞素。如果按照步骤2.3可选的主培养:（488 nm，发射绿色浓度:1激励500），在上述培养:可选包含一个二级抗体，山羊抗豚鼠。 注意:第二抗体，将需要仅如果执行了可选的初级抗体染色（步骤2.3）。为了可视化生物胞素，一STRE建议用在红色发射光谱ptavidin -荧光团共轭，因为它有助于在细节和与更好的对比度（ 图1 - 3）可视化更细轴突。期间双重或三重免疫染色，以避免相互的第二抗体的交叉反应性，添加二级抗体一前一后以串行方式（抗体的序列添加之间2小时间隔就足够了）。 图3A示出没有可选初级抗体染色加工，并切除之前成像的生物胞素填充细胞。 </li></ol><img alt="图3" src="/files/ftp_upload/54880/54880fig3.jpg" /> 图3. 用biocytin形态和切除的恢复成功之后第二免疫染色。 （A1），共焦一个300微米切片示出一个生物胞素填充单元的树枝状形态的恢复的图像。 （&lt;针对+500和-100 pA的电流注射的神经元STRONG&gt; A2）膜电压痕迹。 （B）安装和成像后切除了300微米的部分（A1）之后获得的50微米的部分生物胞填充SOMA的恢复。 （B2）薄款的后续免疫染色揭示了生物胞填充SOMA（右图）与CCK（中图）的共存。合并后的图像在右侧面板中所示。比例尺:50微米。面板A2转载来自Yu 等人的许可。在按25。 <a href="http://ecsource.jove.com/files/ftp_upload/54880/54880fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> （3天） <ol start="6"><li>洗脑切片的3倍，每次10分钟在0.1M PBS（pH值= 7.4）。 </li><li>以保护荧光并促进再染色在未来，装入秒系统蒸发散在水基的安装介质和密封用透明指甲油盖玻片的边缘。 注:最好是尽早复染切片，以避免在去除从滑动，模具侵染，和组织的脱水指甲油由于从载玻片安装介质泄漏的困难。微生物污染可通过在PBS中使用0.02％叠氮化钠来防止。 注意:叠氮化钠是剧毒和易燃，当它在与水接触。请参考危险化学品的处理和处置的标准作业程序。 （天4 - 7） </li><li>在594和488 nm和在20X 40X或放大，揭示在绿色和红色标记神经化学（CB 1 R）生物胞素充满神经元执行的激发聚焦成像。评估colabeling（ 图2）。 </li><li>设定照相机暴露于小于100毫秒，以避免光漂白和OBTA在整个神经元的轴突和树突状乔木树种的共聚焦图像堆栈。使用共焦图像栈和神经跟踪软件包重建神经元形态。 </li></ol> 3.第二主抗体染色注意:当第二免疫染色步骤可以在第一染色之后进行90天，如果第二初级抗体染色7内进行最佳染色达到 - 10天。 （经过7 - 90天） <ol><li>应用丙酮棉签剥离指甲油脱载玻片。 </li><li>小心取出盖玻片用细尖镊子，把0.1M的PBS几小滴上的章节。 </li><li>仔细用细刷油漆去除幻灯片的部分，并把它们洗干净在0.1M PBS 24 -在4℃48小时的覆盖在低光铝箔振动筛 注:关键是要覆盖一个章节luminum箔避免漂白。 </li><li>以确保完全除去在安装介质，洗部分1 - 2倍于次日在0.1M PBS中，每次10分钟。 </li><li>与孵育继续在第二初级抗体（ 例如，胆囊收缩，在某些GABA能中间（CCK发现的神经肽;浓度:1:1000;鼠单克隆抗体））为1天（ 图2）。 注:此过程类似于步骤2.3，但使用不同的抗体。此外，重新染色过程中省略了阻挡步骤。 </li><li>洗脑切片三次，每次10分钟，在0.1M PBS（pH值= 7.4）。 </li><li>染色用荧光标记的二抗山羊抗小鼠（浓度:1:500;激发波长:647纳米），确保二次抗体的激发发光光谱不与链霉（浓度重叠:1:1000;激发波长594纳米），免疫前污渍。 </li><li>安装在水介质中安装的部分，并用密封透明指甲油盖玻片的边缘。 注:在一个不透明的储物盒在4℃保存的部分，以保护荧光。 </li></ol>

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作者什么都没有透露。

该议定书中的关键步骤 用生物胞素填充修补细胞是确保形态的完全恢复最关键的步骤。用于小区的完全恢复，有必要选择最佳的切片方向切割时，尽量减少进程的切断。这种取向可以有所不同的基础上被检查电路和细胞类型。接着，它是必不可少的，以便有足够时间使生物胞素扩散到树突和轴突。某些染料，如neurobiotin，也能穿透通过间隙连接耦合到记录单元的...

大脑已知在其个别的神经元元件的结构和功能特征的多样性。了解不同的神经元类型的脑功能和病理的作用，需要鉴定和神经元的明确标识。在结构上，由躯体树突位置定义的形态特征决定了一个给定的神经元接收到潜在的输入，而轴突树枝状图案识别潜在的突触后目标。神经元的结构多样性拉蒙以来Ÿ卡哈尔的开创性组织学研究1天一直赞赏。单细胞记录技术的出现表明，结构不同的神经元也显示在射击模式和突触特性的差异。在结构和生理的多样性中GABA能抑制性神经元2,3是特别明显。此外，它已成为日益明显的是，structurallŸ类似的神经元可以表达不同的神经化学标记，并显示相应的功能4分歧。同样地，具有相同的神经化学标记物的神经元可以具有不同的结构和功能:5-10。因此，在实践中，神经元的功能特性在网络中的分析和其职位的职责包括限定两形态和神经化学身份。即使靶向特定的神经化学标记物记者鼠标线的出现，通常有必要基于免疫组织11，以确定形态和亚型的身份。 用于表征记录在急性脑切片的细胞的标准方法是在记录过程中与生物胞素或neurobiotin进行填充，固定在多聚甲醛（PFA）以下的记录的部分中，并用免疫组织化学揭示形态和神经化学。因为节的厚度为切片生理学通常300微米以上，因为大多数抗体失败通过深度穿透一路，切片需要被重新切片至60微米或更小，以允许用于生物胞素和神经化学标记物12-14同时免疫染色。不幸的是，后方交会费时费力;切片过程中组织的风险损失;并且可导致差分组织皱缩，复杂形态重建。此外，形态的现有知识能帮助缩小候选标记有可能被细胞中表达。我们已修改标准生物胞素免疫组织学协议，以允许部分的串行处理首先为形态的恢复，然后为潜在的神经化学标记物的鉴定。 免疫组织化学是在组织或细胞的抗原分布的研究，可以使用酶，荧光标记，放射性元素，或金胶体颗粒15被可视化。我的程序nvolves用初级抗体特异性标记和扩增一个或多个特定的抗原，随后通过使用靶向用于可视化的第一抗体荧光第二抗体。由于需要区分各二次抗体的荧光光谱没有重叠，可以同时检查只有抗原的数量有限。因此，形态学的先验知识可以是在选择候选的神经化学标记物对细胞分类是有用的。从概念上讲，已经染色切片的串行处理背后的基本原理是基于免疫标记为一种蛋白质或肽不应与抗原性和随后的免疫标记干扰要结构上独立的肽16的前提。这种缺乏干扰是由于结合的抗体与抗原上的特定的蛋白质的表位，并因此允许多个抗原在相同组织中同时染色。抗原数reveale通过免疫染色d由需要对荧光第二抗体的非重叠光谱和所需要的目标，在不同物种中产生的抗体个体的抗原，以便消除交叉反应性17,18限定。虽然这是后面串联而非同时标记推理与可能相互作用，据我们所知，免疫标记对安装部分的一个或多个抗原的完成后尚未见报道免疫染色第二抗原两个不同的抗体。在这里，我们描述了预先染色和安装部的串行免疫染色的方法。而我们详细介绍此过程为形态的，随后通过染色在厚的部分蛋白质/肽标记恢复的串行免疫标记过程中，相同的程序可在标准中使用，薄的组织切片，以及。此外，我们描述了一个切实可行的办法，以填补记录神经元生物胞素和撞出的过程从记录的结束时的电池用电极，以优化轴突和神经元的树突乔木的填充，如在我们最近的工作6,8-呈现。 此处描述的过程中最关键的优点在于，所记录的细胞的形态可完全恢复并且试图切除或免疫染色切片前成像。虽然与某些抗体的渗透问题可能会导致有必要切除切片进行二次免疫染色，这里详述的程序将消除需要重建从多个部分复杂的神经元，并能避免由于组织损失，差示收缩的问题，这可能会危及重建以下切除。一个额外的优点是，该过程将通过限制免疫染色和重新切片，其中生物胞素填充神经元被回收切片降低成本，时间，精力和昂贵的抗体。最实际的方面是广告可在部分染色月使用上述技术之前进行ditional免疫。特别是，形态学的回收将大大减少从细胞生理数据由于无法获得细胞类型的基本形态特征丢弃的可能性。

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">NaCl&#160;</td>
			<td style="width:211px;">Sigma</td>
			<td style="width:203px;">&#160;S7653</td>
			<td style="width:151px;">Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCL&#160;</td>
			<td>Fluka</td>
			<td>60129</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2HPO4&#160;</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4&#160;</td>
			<td>Sigma</td>
			<td>229806</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Guinea pig anti CB1&#160;</td>
			<td>Sigma</td>
			<td>Af530-1</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CCK</td>
			<td>CURE, UCLA</td>
			<td>courtesy of G. Ohning</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Parvalbumin</td>
			<td>Swant</td>
			<td>PV27</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin, Alexa Fluor conjugate</td>
			<td>Molecular Probes</td>
			<td>S11227</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectashield&#160;</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibodies</td>
			<td>Invitogen Molecular probes</td>
			<td>Alexa Fluor conjugated dyes</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labnet orbit low speed shaker</td>
			<td>Bioexpress</td>
			<td>S-2030-LS</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Dumont</td>
			<td>11231-30</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide folders</td>
			<td>EMS</td>
			<td>71520</td>
			<td>Immunostaining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome VT 1200 S</td>
			<td>Leica</td>
			<td>14048142066</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiclamp 700B amplifier</td>
			<td>Molecular devices</td>
			<td>Multiclamp 700B</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCLAMP 10 Software</td>
			<td>Molecular devices</td>
			<td>pCLAMP 10&#160;</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitizer</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440 digitizer</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter tips</td>
			<td>Nalgene</td>
			<td>171-0020</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15-335-100</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microloaders</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>Electrophysiology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biocytin</td>
			<td>Sigma</td>
			<td>B4261</td>
			<td>Electrophysiology</td>
		</tr>
	</tbody>
</table>

immunostaining of biocytin-filled and processed sections for neurochemical markers

使用膜片钳技术的细胞电生理记录已经允许基于放电模式不同的神经元的类型的识别。生物胞素/ neurobiotin在记录电极列入允许的形态细节，这是必要的，以确定树突分支和由记录神经元的轴突定位的区域事后恢复。然而，鉴于形态相似的神经元的存在下与不同的神经化学特性和功能，细胞类型特异性的蛋白免疫组织化学染色是必不可少的明确识别神经元。为了保持网络连接性，对于生理记录脑切片以厚度为300微米或更大的制备。然而，该厚度常常阻碍免疫组织后处理由于与抗体渗透的问题，因此需要的组织的切除。片切除是一个具有挑战性的艺术，往往resul亭在组织和细胞的形态的损失，所得到的电数据，使该数据无法使用。由于形态将限制在神经元标记的选择数据丢失和引导的恢复，我们采用先恢复细胞形态的策略，其次是二次免疫。我们介绍一个实用的方法，生物胞素中的生理记录和形态的恢复随后连续染色，其次是部分的restaining确定神经化学身份尽显无遗。我们报告，即充满了生物胞素的部分，用低聚甲醛固定（PFA），染色，并盖上盖玻片可以移除并用第二初级抗体天后复染。此restaining包括去除盖玻片，切片在缓冲液中洗涤，并一次和二次抗体的孵育以显示神经化学身份。该方法是用于消除的DAT有利由于无法收回的形态和用于缩小的神经化学标记物的损失，以基于形态学进行测试。

成功完成后，将切片保持在步骤2中进行的生物胞素填充物和免疫标记，并且可以使用共焦或荧光显微镜成像。此外，经处理的部分也将显示免疫染色对在步骤3中随后处理期间标记在图2中示出的部分中的抗原，在一个厚部（300微米）一个生物胞素填充神经元的形态，使用链霉抗揭示在红色和第一主（CB 1 R）的可视化可视绿色。第一次免疫染色PFA固定的...

Watch this Scientific Journal Video about Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers at JoVE.com

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

该协议提出了在使用生物胞素填充和随后的免疫组织化学后处理电生理记录修补神经元的形态回收的方法。我们表明，该染色和盖片厚生物胞素填充部分可以与第二初级抗体天或数月后进行复染。

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

immunostaining-biocytin-filled-processed-sections-for-neurochemical

Research

JoVE Journal

Neuroscience

22.9K 次观看.  Rutgers New Jersey Medical School. 该协议提出了在使用生物胞素填充和随后的免疫组织化学后处理电生理记录修补神经元的形态回收的方法。我们表明，该染色和盖片厚生物胞素填充部分可以与第二初级抗体天或数月后进行复染。 

视频: Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2&#956;m thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.

Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 &#181;m sections with toluidine blue

甲苯胺蓝染色树脂嵌段评价周围神经形态学

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...