The authors would like to thank Mr. Ryan Hart and Mr. Arriz Lucas for their invaluable feedback on this communication. This work was supported by NIH NINDS R01 NS065052 and Phoenix Children&#8217;s Hospital Mission Support Funds.

注：所有的程序，进行了符合亚利桑那大学的机构动物护理和使用委员会（IACUC）。推荐材料和设备的列表可以在表1中可以找到。 1.组织准备<ol><li>灌注<ol><li>安乐死啮齿类用戊巴比妥钠（25毫克/千克，IP）的过量，并用磷酸盐缓冲盐水（PBS），直到完全放血（3-5分钟）以8毫升/分钟的流速灌流transcardially。为深入灌注说明，请参阅量具等2012 9。 </li><li>紧接的PBS冲洗，通过用在PBS中的4％多聚甲醛以8毫升/分钟的流速灌流15-20分钟修复组织。 </li><li>除去脑并置于4％多聚甲醛24小时，随后经分级蔗糖溶液（15％，30％，30％，在序列;在Tris缓冲盐水中制备的）在4℃下。大脑转移到随后的蔗糖溶液0NLY大脑后已经沉没在每个解决方案。注意：通常，5天中的各溶液是足够的时间进行组织下沉。 </li></ol></li><li>组织冻结和Cryosectioning <ol><li>将脑在包埋介质中，例如OCT化合物中，并在-35℃的温度下浸没在异戊烷。让大脑冻结为至少10分钟，然后储存在​​-80℃。可能会出现问题，如果不采取勤奋温度;请参阅讨论的故障排除信息。 </li><li>切串行冠状切片，厚度为20μm和-20℃的温度下进行。收集到的组织正电荷的幻灯片。脑切片可以被放置在一个滑动箱用铝箔包裹在一个拉链袋中并存储长期在-80℃。存储此方法创建一个双重边界，以防止暴露于空气和霜冻。 </li></ol></li></ol> 2.组织处理注：例如设备和材料研究equired染色示于图3中 。替代是可用的，然而，这些图像将有助于这些新的免疫组织化学染色，以在购买之前可视适当的项目。 <img alt="图3" src="/files/ftp_upload/52293/52293fig3highres.jpg" /> 需要实施例的物品为免疫组织化学染色的黑盒子中的 （A）示出的是一种理想的湿度室中进行免疫荧光，如载玻片避光，而不需要包裹上金属箔的框：图3。以下切片，幻灯片可以存储在一个盒子如黄箱中的（B）所示。紧紧包裹箱箔和冷冻前放置在一个拉链顶袋有助于保护组织样本从冷冻烧伤。幻灯片的示例（C）中，与（D）中示出不同的染色皿和给出（E </strong&gt;）。盖玻片可以在尺寸和厚度（F）各不相同，但1.2厚的盖玻片提供最正直和共聚焦显微镜不错的成像效果。铅笔诸如在（G）中所示，可用于标记的幻灯片。永久标记应避免作为油墨可以运行，既影响染色，并确定所述样品是什么的能力。迷你笔PAP如在（H），使显示的幻灯片上要绘制的边界驱蚊。 <ol><li>幻灯片准备<ol><li>在室温下删除冰柜和解冻的幻灯片。 <ol><li>可选：如果部分已经从幻灯片先前浮起，放置解冻载玻片在烘箱中在60℃下不超过4小时，以帮助防止组织切片从浮脱幻灯片。 </li></ol></li><li>地方幻灯片在幻灯片机架以及相应的菜。 </li><li>洗涤滑入PBS洗3次，每次5分钟，改变洗涤之间溶液。从这一步，避免secti组件没有液体延长的时间周期。注意：如果切片干燥，背景染色增加并且有意义的数据不能可靠地获得。 </li></ol></li><li>组织染色<ol><li>在一个不透光的盒子染色，营造“湿室”与浸泡用去离子水无绒毛组织。 </li><li>干燥，不起毛的组织幻灯片的边缘，使用mini PAP笔做出拒液边框在幻灯片的边缘，远离组织切片。该边界应确保液体的弯液面，并在组织，使得表面张力不影响染色的边缘之间充足的空间。 注：巴氏笔驱蚊边框可以应用之前2.1.3如果感兴趣的抗体并不需要微波抗原修复。如果巴氏笔之前已经在PBS中洗涤应用，则疏液边界的完整性必须在此步骤进行检查。使用一个微型的PAP笔填写在边境的任何空白。 </li><li&gt;用幻灯片水平放置，阻断非特异性抗原孵育在4％V / V血清的PBS（块解决方案）的结合。吸管300微升，在室温下每滑块溶液1小时。确保块溶液延伸至巴氏笔在滑动的边缘，并完全覆盖所述组织，以避免造成邻近组织表面张力不均匀的染色。 <ol><li>从其中的二级抗体是由相同的物种使用血清。注意：在此过程中，二次抗体在驴制成，因而驴血清被使用。如果来自两个或更多不同物种的第二抗体的使用，包括血清来自每个物种。 </li></ol></li><li>吸取第一抗体在载玻片上。注：抗体浓度的这种染色进行了优化，以1：5000和1：500分别为Iba1和泛神经元。这些浓度已经被发现显示出有意义的染色用无背景染色。 <ol><li>稀释块解决方案在PBS 1％血清和添加第一抗体。吸取300微升的主要抗体溶液中每张幻灯片的1％血清。再次，确保流体的巴氏笔的边缘。孵育过夜，在4℃。 </li><li>包括三个控制幻灯片：一个包含既不Iba1也不泛神经元抗体，其中一个Iba1不泛神经元抗体，和一个与泛神经元抗体没有Iba1。在相同的运行具有相同的溶液染色这些幻灯片，但是省略了初级抗体，以测试非特异性次级抗体的结合。 </li></ol></li><li>第二天早晨，洗涤滑入PBS洗3次，每次5分钟，改变洗涤之间溶液。 </li><li>荧光抗体对光敏感，因此，从这个进步，最大限度地减少曝光，确保清洗容器包装在铝箔和杂交框是黑色或避光培养。吸取适当的二级抗体上的所有幻灯片和孵育60分钟在室温下以1:1的浓度：在方框溶液250（见步骤2.2.3）在一个不透光的“湿度箱”（见步骤2.2.1）。 <ol><li>使用不同波长的次级抗体。这里，对于第一抗体兔抗Iba1，用驴抗兔594作为相应的二次抗体。为第一抗体的小鼠抗泛神经元，使用驴抗小鼠488作为相应的二次抗体。或者，使用抗兔488和抗小鼠594。 </li></ol></li><li>洗涤滑入PBS洗3次，每次5分钟。 </li><li>可选：执行核染色。 <ol><li>地方在赫斯特（或其他核染色）以0.03微克/毫升双蒸H 2 O中的浓度为恰好60秒。 </li><li>洗涤滑入PBS洗3次，每次5分钟。 </li></ol></li><li>在双蒸2 0洗。 </li></ol></li><li>盖片<ol><li>盖玻片载玻片用含水封固剂，如氟omount-G或ProlongGold。小心除去用棉签所有的泡沫。 注：其他的安装剂可以使用，但是高的渗透染料之间已注意到一些内盖片的天。 </li><li>用透明指甲油密封边缘，防止部分变干，由于蒸发。允许指甲油干燥在一个不透光的容器中，同时滑动保持平坦，在室温下，然后存储在一个不透光的容器用铝箔包裹在4℃。 </li></ol></li></ol> 3.影像学检查染色组织<ol><li>显微镜<ol><li>让指甲油开始显微镜，这应该发生在一个黑暗的房间前干燥至少一小时。 </li><li>获得使用共焦或研究显微镜用荧光光源和数字照相机附件的显微照片。使用附带的软件，设置曝光每个波长 - 405，488，594 - 分开。注：深入成像指令应可在网上从显微镜制造商。 </li><li>获得在每个通道中的显微照片，而无需移动部分或调整焦点。取的图像的颜色，或交替地以灰度，并转换为颜色之后。 注意：颜色或灰度图像的每个通道都可以在后期处理进行整理。 </li><li>确保组织切片不暴露于环境光或显微镜光为长时间，如将发生光致漂白的部分。为了避免这种情况，增加曝光时间，而不是增加光/激光强度。 </li><li>不内被接通30分钟关掉荧光光源。 注意：切换源通断在快速连续可能降低荧光灯泡的寿命。 </li></ol></li></ol>

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The authors have nothing to disclose.

这种沟通的总体目标是引进免疫程序给读者。为此，双标记与Iba1和泛神经元抗原的例子来观察小胶质细胞和多聚甲醛中的神经元灌注，蔗糖冷冻保护，冰冻切片大鼠脑使用。 这种技术可以适用于服务无休止的目的。不同抗原在多种组织类型，例如但不限于脑，肺，肝，肾和肠的阵列可以免疫组织化学与小幅调整协议被可视化。免疫组织化学允许的多个标记的直接比较，并且?...

<html>免疫组织化学是用于通过使用特异性结合到感兴趣的抗原的第一抗体检测抗原（ 即蛋白）的组织切片的方法。免疫组化法开创的JR马拉克在1934年，当他确定抗体可与很大的特殊性1本地化抗原。在1942年开始，一些先在体外用荧光抗体可视化免疫研究发表2,3，此后，第一体内组织化学研究发表4。在20世纪60年代，经过三十年的免疫组化方法以来，酶标记的抗体开始被用作辅助试剂。这些方法同时并独立开发了法国和美国5,6。如今，抗体的广泛提供了无限的可能性，免疫组化研究7。 “&gt;此对应的总体目标是提供一个简单的介绍到免疫组化染色;它并不意味着是该技术的全面和详尽的审查中列出的方法，免疫组化技术对两种抗原呈现（标记为小胶质细胞和。神经元），用于多聚甲醛的染色灌注，蔗糖冷冻保护，冰冻切片大鼠脑。免疫组织化学染色开始阻断非特异性抗原结合，以减少背景染色。接着，温育与初级抗体允许在组织结合到特定抗原。继第一抗体，另一种抗体，称为二次抗体，被施加到第一抗体链接到一个共轭可视信号8。二级抗体靶向免疫球蛋白G（IgG）的特定于其中初级抗体升高的物种域。二级抗体放大该信号第一抗体的自吨的Fab区他的第二抗体结合到多个站点上的第一抗体的抗体结构域。或者缀合到二级抗体的F c的区域的酶或荧光分子使可视化。例如，兔抗Iba1第一抗体是特异性针对Iba1兔IgG分子。当驴抗兔IgG被作为二次抗体施加，它会识别和结合的兔抗Iba1 IgG抗体的多个区域（ 见图1）。驴抗体可通过各种方法来显示。这种对应着重于检测缀合到二级抗体，其识别第一抗体，用于可视化通过荧光显微镜荧光团。在荧光免疫组织化学，核染色剂如Hoechst的或DAPI可以用于可视化的所有核。 <img alt="图1" src="/files/ftp_upload/52293/52293fig1highres.jpg" /> 图1：SCH的直接与间接的抗体标记技术ematic表示。抗体结合到感兴趣的抗原，并且可以通过产生针对第一抗体的物种的二级抗体进行放大。这种技术可以使用抗生物素蛋白-生物素复合物（ABC）用于扩增和DAB进行可视化（A）中进行，或直接缀合的荧光第二抗体（B）。另外，主要的抗体可直接偶联与许多不同的标记，包括生物素或荧光团（C）。 <a href="https://www.jove.com/files/ftp_upload/52293/52293fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 对于免疫组织化学染色的可视化的另一种方法是使用3,3&#39;-二氨基联苯胺四盐酸（DAB;参见图1和2）。这不同于荧光通过使用生物素化的或辣根过氧化物酶（HRP）缀合的第二抗体，其提供了一种酶的DAB转换为沉淀物是在亮视野显微镜可见。中的实例，其中一个单一的抗原是感兴趣或染色需要是持久的，DAB可能比荧光染色更合适。然而，DAB染色并不非常适合于多个标记之间的差异，特别是如果两个核抗原的兴趣。关于DAB材料和协议修改的信息，请参阅表1中 。另外，硝基蓝四唑氯化物/ 5-溴-4-氯-3-吲哚磷酸（NBT / BCIP）可以被用于可视化的碱性磷酸酶（AP）缀合的二抗体。 <img alt="图2" src="/files/ftp_upload/52293/52293fig2highres.jpg" /> 图2：镍增强的DAB单标记的大鼠脑组织切片的代表性图像大鼠脑瑟ctions其上标有镍增强DAB的Iba1（A）和泛神经元（B）允许单独或小胶质细胞神经元的长期分析。比例尺20微米。 <a href="https://www.jove.com/files/ftp_upload/52293/52293fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 必须考虑的组织内感兴趣的抗原的估计的丰度的分析处理。间接的方法（如上所述）可用于具有低丰度的目标是有用的。当感兴趣的抗原是在高丰度，直接的方法都可以应用。直接方法涉及一个直接缀合到一个可视信号的初级抗体，因此没有第二抗体是必需的。此方法简化了染色处理，但消除了间接的方法取得的扩增。使用直接缀合的初级抗体也消除交叉反应的第二抗体的当双标记。 该通信详细介绍了协议双标记与Iba1和泛神经元（ 如表1的细节）。 Iba1小胶质细胞染成许多活化状态，包括分枝，超分枝，激活，变形虫，和杆。泛神经污渍神经轴突，树突和索玛。因为Iba1渍最小胶质细胞和泛神经元靶神经元，污渍的这种组合中获得的小胶质细胞 - 神经元相互作用的广泛了解。 总之，免疫组化染色依赖于精心挑选的抗体。随着研究的问题变得更加具体，提出的到备用抗原抗体可能不理想。要针对特定​​的小胶质细胞激活状态，可以选择使用CD45或CD68抗体，而不是Iba1。此外，与小鼠的工作，F4 / 80可提供必要的结果。同样地，神经元元件可特异性地与抗体ラ针对性反对核ISED，突触（前或后），轴突和生长锥。此外，还有其他的标志物区分神经元（双肾上腺皮质激素，的NeuN）的年龄和神经再生（GAP-43）。

<table border="0" cellpadding="0" cellspacing="0" width="1171">
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			<td height="21" style="height:21px;width:399px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;">Fisherbrand Superfrost Plus Glass Slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>22-034-979</td>
			<td>Used for tissue mounting (1.2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Oven</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:119px;">51028112</td>
			<td>Used for tissue drying (2.1.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mini Pap pen</td>
			<td style="width:132px;">Life Technologies</td>
			<td style="width:119px;">00-8877</td>
			<td>Used in step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Andwin Scientific Tissue-tek Slide Staining Dish</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">22-149-429</td>
			<td>Used for all washes during staining (2.2), as well as the Hoechst step (2.2.8)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Kimwipes</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">06-666-A</td>
			<td>Used for drying slides (2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Black Staining Box</td>
			<td style="width:132px;">Ted Pella</td>
			<td style="width:119px;">21050</td>
			<td>Used for blocking and staining steps (2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Normal Donkey Serum</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">50-413-253</td>
			<td>Used for block and antibody incubation (2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse &#945;-Pan-neuronal</td>
			<td style="width:132px;">Millipore</td>
			<td style="width:119px;">MAB2300</td>
			<td>Used for primary antibody (2.2.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rabbit &#945;-Iba1</td>
			<td style="width:132px;">Wako Chemical</td>
			<td style="width:119px;">019-19741</td>
			<td>Used for primary antibody (2.2.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Donkey &#945;-Rabbit 594</td>
			<td style="width:132px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">711-585-152</td>
			<td>Used for secondary antibody (2.2.6)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Donkey &#945;-mouse 488</td>
			<td style="width:132px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">715-545-150</td>
			<td>Used for secondary antibody (2.2.6)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Caterer&#39;s foil</td>
			<td style="width:132px;">Any</td>
			<td style="width:119px;">N/A</td>
			<td>Used in steps 1.2.2 and 2.3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fluoromount-G</td>
			<td style="width:132px;">Southern Biotech</td>
			<td style="width:119px;">0100-01</td>
			<td>Used for coverslipping (2.2.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Coverslips</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">12544E</td>
			<td>Used for coverslipping (2.2.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Clear Nail Polish&#160;</td>
			<td style="width:132px;">Any</td>
			<td style="width:119px;">N/A</td>
			<td>Used for coverslipping (2.2.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Axio Observer.Z1 and LSM 710 (laser scanning, confocal)</td>
			<td style="width:132px;">Carl Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td>Used for imaging (3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Axioskop A2</td>
			<td style="width:132px;">Carl Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td>Used for imaging (3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CitriSolv&#160;</td>
			<td>FisherScientific</td>
			<td></td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABC</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAB Peroxidase kit</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated horse &#945;-rabbit IgG</td>
			<td>Vector Laboratories</td>
			<td>BA-1100</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated horse &#945;-mouse IgG</td>
			<td>Vector Laboratories</td>
			<td>BA-2001</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Hydrogen Peroxide</td>
			<td>FisherScientific</td>
			<td>H325-500</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Wheaton slide racks and staining dishes</td>
			<td style="width:132px;">TedPella</td>
			<td style="width:119px;">21043</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex perfusion pump and tubing</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>Used for perfusion (1.1.1 and 1.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Andwin scientific tissue-tek CRYO-OCT compound (case of 12)</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td>Used for tissue freezing (1.2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Thermometer (-50 to 50 C)&#160;</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">15-059-228</td>
			<td>Used for tissue freezing (1.2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica&#160;</td>
			<td>CM3500S</td>
			<td>Used for tissue sectioning (1.2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staining Dish, Plastic with 2 Lids</td>
			<td>Grale Scientific</td>
			<td>353</td>
			<td>For antigen retrival</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 Place Staining Rack, Slides Horizontal</td>
			<td>Grale Scientific</td>
			<td>354</td>
			<td>For antigen retrival</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microwave</td>
			<td>Any</td>
			<td>N/A</td>
			<td>For antigen retrival</td>
		</tr>
	</tbody>
</table>

primer for immunohistochemistry on cryosectioned rat brain tissue: example staining for microglia and neurons

免疫组织化学是一种广泛使用的技术，用于检测的存在，位置和相对原位抗原的丰度。这个入门级协议说明了试剂，设备和完成啮齿动物的脑组织的免疫组织化学染色所需的技术，使用标记的小胶质细胞和神经元的元素作为一个例子。具体地，本文是一步一步的协议，用于小胶质细胞和神经元的通过免疫组织化学对Iba1和泛神经元，分别荧光可视化。荧光双标记是用于在同一样品中多种蛋白的定位是特别有用的，提供的机会，以准确地观察细胞类型，受体，配体之间的相互作用，和/或细胞外基质中相对于彼此以及蛋白质共单个细胞内的定位。不像其他的可视化技术，荧光免疫组化染色强度可能减小在几个星期到几个月之后染色，除非有适当的预防措施。尽管这种限制，在许多应用荧光双标记优于替代品，如3,3&#39;-二氨基联苯胺四盐酸盐（DAB）或碱性磷酸酶（AP），由于荧光是更多的时间效率，并允许两个或更多个之间更加精确的分化标记。讨论包括故障排除提示和建议，以促进成功。

这在大鼠的脑组织切片染色方案的结果已小胶质细胞的荧光标记，在594通道（红色）和神经元标记中的488通道（绿色;参见图4）。如果一个核染色已经完成，它将显示在405通道（蓝色）。图像可以采取不同的通道和覆盖该三个信道的直接比较，或任何两个通道之间。许多数字采集软件套件包含此功能。双标记与这里显示Iba1和泛神经元标记表明大部分分枝小胶质细胞，长细神经元的预?...

Watch this Scientific Journal Video about Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons at JoVE.com

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织：例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

primer-for-immunohistochemistry-on-cryosectioned-rat-brain-tissue

Research

JoVE Journal

Neuroscience

31.6K 次观看.  University of Arizona College of Medicine - Phoenix. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example. 

视频: Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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 ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

应用 ImageJ 对免疫组化制备组织显微照片小胶质细胞形态学的定量研究

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.