This work was supported by the Alzheimer&#8217;s Association (NIRG-10-171721; to U.H.), National Institute of Mental Health (MH096702; to U.H.), National Institute of Neurological Disorders and Stroke (NS081333; to C.M.T.), and pilot study awards from the National Institute on Aging-funded Alzheimer&#8217;s Disease Research Center at Columbia University (AG008702; to J.B. and Y.Y.J.) that also supports the New York Brain Bank. We thank members of the Hengst laboratory for comments and discussions

所有动物的程序批准了哥伦比亚大学的IACUC和护理和使用实验动物适用的准则随访。注意：准备用于在无RNase或DEPC处理水的ISH程序的所有缓冲区。这个建议并不是严格必要的ISH已经完成之后，但它被认为缓冲器仍然制备蒸压双蒸水和/或通过过滤消毒。 1.检测ATF4基因定位于胆碱能轴突使用荧光成年小鼠大脑中的原位杂交（FISH），其次是免疫组化<ol><li>对于多聚甲醛固定冷冻大脑切片样品制备<ol><li>在下面的例子，从准备冷冻固定老鼠大脑切片。 <ol><li>简单地说，牺牲9个月大的小鼠麻醉氯胺酮（100毫克公斤-1体重）和甲苯噻嗪（10毫克公斤-1身体weigHT），随后加入4％多聚甲醛的PBS穿心输注（pH 7.4）中。 </li><li> 24小时在4℃取出大脑和定位后在4％多聚甲醛</li><li>固定后，24小时在4℃洗涤样品在PBS洗三次并cryoprotect在30％蔗糖的PBS（pH 7.4）中</li><li>在OCT中嵌入大脑冷冻化合物，串联部分中，在20微米在恒冷切片机和安装在聚-D-赖氨酸处理的显微镜载玻片。保持大脑切片在-20℃直到使用。 </li></ol></li><li>除去从冰柜和风干多聚甲醛固定的脑切片在室温30分钟。 </li><li>用1×PBS洗涤三次。 </li><li>在通风橱中，重新修复脑切片20分钟，用4％多聚甲醛在室温。 注：警告：4％多聚甲醛是有毒的，必须被处理和丢弃的适当以下安全协议。 </li><li>用1×PBS洗涤三次。 </li><li>脱水的大脑切片。 <ol><li>制备50％，75％和100％乙醇溶液。 </li><li>沉浸在50％乙醇载玻片5分钟，室温。 </li><li>沉浸在75％的乙醇载玻片5分钟，室温。 </li><li>沉浸在100％乙醇玻片5分钟，室温。 </li><li>沉浸在新鲜的100％乙醇载玻片5分钟，室温。注：或者，幻灯片可以存储在100％的乙醇，在-20℃下长达1个月。 </li></ol></li></ol></li><li> FISH检测其次是利用免疫组织化学热诱导抗原/ RNA揭露<ol><li>在开始之前准备好所有必需的材料： <ol><li>热火杂交炉至40℃，将装有蒸馏水的盘在烤箱创造湿润的环境。 </li><li>取一个滑动箱并放置浸泡在蒸馏水内加湿滑动箱纸巾。放置在杂交炉滑动箱。 </li><li>从冰箱中取出探针（消极和靶探针），在杂交烘箱加热它们10分钟。让探针冷却道北，RT。 </li><li>平衡的扩增试剂的AMP 1-4（包括在荧光复合试剂盒）于RT。 </li><li>制备抗原修复液：10mM柠檬酸钠（pH值为6），0.05％吐温20。注意：溶液可以储存在室温无限期和用于将来的染色。 </li><li>制备1×洗涤缓冲液稀释在无RNase或DEPC处理水的50倍原液（包含在荧光复合试剂盒）。 注：1×洗涤缓冲液可以储存在室温下1个月，用于将来的染色。 50X洗涤液可能沉淀。如果是这样，在制备1×溶液之前40℃下进行10分钟热的50x洗涤缓冲液。 </li></ol></li><li>除去从100％的乙醇和风干载玻片5分钟，室温。 </li><li>制备含有50毫升抗原修复液的科普林缸。沉浸在抗原修复液的幻灯片和煮沸他们在微波炉中加热10分钟。 注：或者水平的地方在滑动100毫米直径的圆玻璃盘含有100ml检索溶液。 <ol><li>填满的1L烧杯用蒸馏水，并将其放置在微波炉。 注：执行时，揭露水会“缓冲”的热量。 </li><li>放置在微波炉内抗原修复溶液的幻灯片。煮沸滑动以高功率5分钟。样品已停止沸腾后，立即再次加热幻灯片在高功率额外的5分钟。 </li><li>冷静下来的幻灯片在RT。 注：关键步骤：沸腾持续时间和过程应该由用户来确定，这取决于微波特性。越快沸腾开始越高溶液蒸发的可能性。在这种情况下，建议样品煮沸的时间较短的两倍以上，完成10分钟共揭露时间。相反，如果在中等或低功率进行加热，揭露可进行10分钟进行一次。但是，如果样品不开锅continuously代表总共约10分钟，这可能会导致无论是靶RNA和蛋白质部分去掩蔽被检测到。 </li></ol></li><li>洗滑动三次用1×PBS。 </li><li>添加2 - 3滴dapB的探头设置了用于评估背景染色的脑片（约100微升）。注：探头的dapB设定的目标细菌的基因，不应该承认任何哺乳动物RNA。 </li><li>添加2 - 3滴靶向探针设置为用于检测目的RNA的那些脑片。注意：一探针组靶向残基20 - 1381鼠标ATF4的mRNA是用在本实施例的。 </li><li>盖玻片用封口膜，以避免探头挥发，将它们放置在杂交炉内部的加湿盒滑动，并培育它们在40℃2小时。 注：可选：额外的大脑切片可以用杂交探针一套针对鼠标和Polr2A作为阳性对照。 </li><li>洗SLI德两次在室温2分钟1×洗涤缓冲液。 </li><li>添加2 - 3滴扩增试剂AMP 1-FL到每个脑切片，盖玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉30分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>添加2 - 3滴扩增试剂AMP 2-FL到每个脑切片，盖玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉15分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>添加2 - 3滴扩增试剂AMP 3-FL到每个脑切片，盖玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉30分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>添加2 - 3滴扩增试剂AMP 4-FL到每个脑切片，盖玻片用封口膜，将它们放置在滑动箱和孵育在40℃下在杂交炉15分钟。 </li><li>洗涤用1×洗涤缓冲液进行2分钟，在RT和两次用1×PBS滑动两次。 </li><li>添加100 - 200微升（或足够量，以完全覆盖片）的封闭液含有3毫克/毫升BSA，100mM的甘氨酸和0.25％的Triton X-100的PBS（pH7.4）中的幻灯片，覆盖它们用封口膜和孵育在室温30分钟。 </li><li>添加100 - 200微升抗胆碱抗体稀释于封闭液（1/100）的幻灯片，用封口膜覆盖它们，并培育2天，4℃。确保脑片不干燥。 24小时培养后，如果需要，再申请抗胆碱抗体溶液大脑切片。 </li><li>洗涤滑动在1×PBS清洗3次，每次5分钟，在RT。 </li><li>添加100 - 200微升适当的Alexa标记的二抗的（ 例如 ，的Alexa-594驴抗山羊为这个特定的例子），以幻灯片，盖用封口膜和incub吃1小时，在室温。 </li><li>洗涤滑动在1×PBS清洗3次，每次5分钟，在RT。 </li><li>洗涤滑动一次用蒸馏水。 </li><li>滑山含DAPI安装介质。 </li><li>可视化在荧光显微镜下的脑切片。 </li></ol></li></ol> 2.检测ATF4 mRNA的本地化的轴突在使用显色原位杂交（CISH），其次是Luxol快速蓝和甲酚紫复染人脑样本<ol><li>对福尔马林固定的石蜡包埋的人脑样品的样品制备<ol><li>烘烤片在干燥炉中在60℃1小时。 </li><li> Deparaffinize脑切片在通风橱中。 注：注意：试剂是有毒的，必须进行处理并丢弃以下相应的安全准则<ol><li>沉浸在二甲苯替代清除剂载玻片两次，每次10分钟。 </li><li>沉浸在100％乙醇载玻片两次，持续1分钟。 </li><li>一个IR-干切片5分钟在室温。注：另外可能的样品在室温Ø干燥/ N，但必须在24小时内使用。 </li></ol></li></ol></li><li>二氨基联苯胺（DAB）为主色ISH检测之后luxol快速蓝色和甲酚紫染色用热诱导和蛋白酶引起的RNA揭露<ol><li>在开始之前准备所需材料： <ol><li>热火杂交炉至40℃，并放置装有蒸馏水创造湿润的环境，一个托盘。 </li><li>取一个滑动箱并放置浸泡在蒸馏水内加湿滑动箱纸巾。放置在杂交炉滑动箱。 </li><li>制备1×预处理2稀释在无RNase或DEPC处理水10倍的原液（包含在CISH试剂盒）。如果预处理的热板上进行，加入100毫升的预处理溶液，以100毫米直径的玻璃培养皿，以增加盘与热板之间的接触表面（如果预​​处理在微波中进行，可以使用一个科普林缸代替如在1.2.3规定）。散热解决方案，以100℃，并保持样品淹没煮沸前不超过30分钟。 </li><li>热消极和积极的探头在40℃10分钟，冷却在室温。 </li><li>平衡的扩增试剂的AMP 1-6（包括在CISH试剂盒）于RT。 </li><li>制备1×洗涤缓冲液稀释在蒸馏水中的50x贮备液（包含在CISH试剂盒）。注：1×洗涤缓冲液可以储存在室温下1个月，用于将来的染色。 </li></ol></li><li>加〜4滴预处理1的（〜120微升）到每个脑切片，盖上封口膜和在室温下孵育10分钟。 </li><li>在新鲜蒸馏水洗涤3至5次。 </li><li>进行热诱导的揭露： <ol><li>转移幻灯片热预处理2（包含在CISH试剂盒）解决方案，并煮沸15分钟。注意：沸腾可以在热板上，确保执行连续煮沸或以下）的规定1.2.3关键步骤微波炉。 </li></ol></li><li>立即玻片转移到蒸馏水冲洗3至5次。 </li><li>洗涤滑动在新鲜的100％乙醇3至5倍。 </li><li>风干切片5分钟，在RT。注：另外切片可干燥O / N。 </li><li>执行蛋白酶诱导揭露： <ol><li>加4滴预处理3（包含在CISH试剂盒）中，用封口膜封住，并在杂交炉保温30分钟，在40℃。 </li></ol></li><li>洗涤滑动在新鲜蒸馏水3至5倍。 </li><li>加4滴dapB的探头设置了用于评估背景染色的脑片。 </li><li>加入4滴所述靶向探针设置为用于检测目的RNA的那些脑片。 注：探针组靶向残基15 - 1256人类ATF4 mRNA的是用在本实施例中。可选：额外的大脑切片可以HYBridized用探针组针对人类PPIB 和用作阳性对照。 </li><li>放置在滑动框并孵育2小时。在杂交炉40℃。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>加4滴AMP 1试剂每个脑切片，盖玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉30分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>加4滴AMP 2试剂每个脑切片，盖上玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉15分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>加4滴AMP 3试剂每个脑切片，盖上玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉30分钟。 </li><li>滑洗两次为1XH缓冲液在室温2分钟。 </li><li>加4滴AMP 4试剂每个脑切片，盖上玻片用封口膜，将它们放置在幻灯片框和孵化，在40℃下在杂交炉15分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>加入4滴AMP 5试剂至各脑切片的，覆盖载玻片用封口膜和孵育在室温30分钟。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>加4滴AMP 6试剂每个脑切片，盖上玻片用封口膜和孵育15分钟，在室温。 </li><li>洗涤用在室温2分钟1×洗涤缓冲液滑动两次。 </li><li>混合欠压1的等体积和欠压2试剂（包括在CISH试剂盒）中，添加〜120微升溶液到每个脑切片，盖用封口膜和孵育10分钟，在室温。 </li><li>洗涤滑动两次在RT 1×洗涤缓冲液2分钟，在蒸馏水中冲洗一次。 注：关键步骤：以下步骤是关键获得最优轴突染液无掩蔽的RNA颗粒的存在。 </li><li>之前执行染液检查明显微镜下感兴趣的mRNA的存在。限定的脑样品中监测泪点的整个染液过程中存在参考区域。 </li><li>在60 摄氏度预热luxol蓝快的解决方案</li><li>在luxol蓝色快滑到位，并培育30分钟，60℃。 </li><li>冲洗几次在蒸馏水中。 </li><li>浸滑动多次在0.05％碳酸锂溶液开始分化。 </li><li>浸在新鲜的75％乙醇滑动两次。 </li><li>在蒸馏水中清洗。 </li><li>检查一个明显微镜下大脑切片。需要注意的是灰质和白质起，应该区分和RNA颗粒为深蓝色/黑色的泪点仍清晰可见。验证轴突开始出现的淡蓝色纤维。 </li><li>重复步骤2.2.28到2.2.32。 Perfor米与孵育快速luxol蓝溶液在10 - 20分钟的步骤，仔细监测染液和RNA颗粒的存在。注意：通常，最佳染液在60取得- 90分钟（总温育时间），但时间可以如果它被怀疑的RNA颗粒可能被luxol快蓝染色（参见图2的例子）被掩蔽被缩短。 </li><li>孵育在甲酚紫溶液载玻片在室温10分钟。 </li><li>在蒸馏水中清洗幻灯片。 </li><li>浸滑动在70％的乙醇5至10倍。 </li><li>通过100％乙醇的3快速变化脱水大脑切片。 </li><li>在通风橱中，清脑切片在二甲苯替代清除剂2分钟，第三时间为5分钟，在RT下温育两次。 </li><li>摩脑片二甲苯为主的永久性封固剂。 </li><li>一个明显微镜下分析样品。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这份报告中，我们描述了轴突本地化ATF4 mRNA的检测采用了高分辨率的ISH技术。这些和以前发表的研究报告表明，该技术是与基于抗体的蛋白质检测的组织中或甚至整个胚胎33兼容。重要的是，它最近已被使用于海马神经元34的树突内检测电弧的mRNA。它也可以与组织学染料对组织染色组合。最后，这是适合于同时检测多个靶RNA 28,30,33。这些结果例证的高分辨?...

轴突mRNA的招聘和本地翻译的轴突能够在一个时间和空间的方式急响应1到胞外刺激。轴突内蛋白合成被最好地理解在神经发育的上下文中，其中它在生长锥行为2-8关键作用，轴突寻路9-11和逆行信令12,13。但到目前为止很少有关于轴突蛋白合成后发育神经元的功能意义时，轴突mRNA和核糖体水平都大大降低14,15。成熟脊椎动物轴突长期以来被认为是不活动的平移16。然而，最近的研究表明，本地翻译病理状态下重新激活在成熟轴突。例如，mRNA的一个子集被招募到再生轴突以下神经损伤和轴突内蛋白质合成所需的这些轴突17的正确再生。此外，集团公顷■证明特异mRNAs被招募到轴突后局部暴露于阿耳茨海默氏病的肽Aβ1-42，和转录因子ATF4的本地翻译需要从轴突传播的Aβ1-42的神经变性影响神经元胞体18。最后，高通量分析已经表明成熟轴突具有更复杂和动态的转录比预期18-21，特别是在病理状况。根据这些研究，一个高度敏感和特异的方法来检测在成年神经系统轴突本地化的mRNA是必要的。 许多关于基因招募和成熟轴突当地的翻译工作已经对培养的神经元被执行。这是特别真实对于转录分析自专门培养方法存在允许从躯体树突隔室18-20轴突的隔离。虽然这些研究都给予宝贵的插件飞行进入成熟轴突当地翻译的作用，问题是否培养的神经元忠实代表情况在体内或mRNA的招聘是轴突以培养条件的一种适应性反应仍然是开放的。很少有研究提供了表达的招聘证据成熟轴突在体内 。例如，所述转录物编码的嗅蛋白在成年感觉神经元22轴突已经检测。含有的β肌动蛋白的mRNA的3&#39;非编码区的转基因被输送到在周围和中枢神经系统的神经元的轴突在小鼠和发育阶段23之后被局部翻译。拉明B2 mRNA定位到视网膜轴突Xenopues蟾蝌蚪和枯竭影响轴突发育后21轴突维修。干扰与细胞色素C氧化IV mRNA的编码的轴突运输改变鼠标的行为24。最后，ATF4的mRNA在成年找到的小鼠和人类大脑中的Aβ1-42的范围内xons诱导神经退行性变18。 高通量转录分析已被证明是有用的，以确定在分离的轴突在体外的mRNA型材但对体内研究的限制，因为在整个组织轴突从未发现在隔离但间杂有神经元细胞体，神经胶质细胞和其它细胞类型。因此，这样的分析必须与确认的mRNA的亚细胞定位的成像技术相结合。 原位杂交（RNA ISH）的RNA可在细胞和组织中的特定RNA序列的检测和可视化。然而，原始的RNA原位杂交测定法是只适合识别的高丰度的RNA 25，这是很少对轴突本地化的mRNA的情况。在过去的十年里越来越多的努力已投入开发新技术，使mRNA的检测在单分子水平<suP&gt; 25,26。例如，Singer和同事开发ISH探针来检测的mRNA在单细胞，包括在5个非重叠的荧光标记的50聚体（详情参见27）。上述技术和此处描述的之间的主要区别是，以后使用20双Z型结构（未线性）探针一般是针对〜1kb的兴趣确保特异性和低背景水平的RNA的区域。探针杂交，然后与最终荧光标记或缀合的酶，使显色反应，前置放大器和功率放大器序列。这些扩增步骤改善信噪比相比其他ISH技术28。在这里，我们将介绍使用RNAscope结合无论是与免疫细胞化学荧光或组织学染料使轴突复染两种协议。这两个协议都适合可视化ATF4 mRNA在成年莫轴突本地化使用和人类大脑。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>custom probe targeting residues 20-1381 of the mouse Atf4 mRNA (NM_009716)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>-</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>custom probe targeting residues 15-1256 of the human ATF4 mRNA (NM_001675.2)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>-</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>negative control probe-DapB</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">310043</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>positive control probe-mouse Polr2A (optional)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">312471</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>positive control probe-human PPIB (optional)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">313901</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">320850</td>
			<td>In situ hybridization kit</td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - BROWN (for chromogenic detection)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">310035</td>
			<td>In situ hybridization kit</td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-ChAT antibody</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Luxol Fast Blue-Cresyl Echt Violet Stain Kit</td>
			<td>American MasterTech</td>
			<td>KTLFB</td>
			<td></td>
		</tr>
		<tr>
			<td>Clearify clearing agent (xylene substitute)</td>
			<td>American MasterTech</td>
			<td>CACLEGAL</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold mounting&#160; medium with DAPI</td>
			<td>Life Technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium</td>
			<td>Sigma</td>
			<td align="right">6522</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of axonally localized mrnas in brain sections using high-resolution in situ hybridization

的mRNA经常局限于脊椎动物轴突和当地翻译所需的轴突寻路或在开发和维护，修理或神经变性postdevelopmental周期分支。高通量分析最近透露，轴突有一个更加动态和复杂的转录于此前预期。这些分析，但大多已在培养的神经元轴突的地方可以从躯体树突状车厢被孤立进行。这几乎是不可能实现这种隔离在体内整个组织。因此，为了验证的mRNA和它们的功能关联的募集在整体动物，转录组分析，最好应与技术，允许mRNA的原位可视化相结合。最近，已经开发出新颖的ISH技术，检测的RNA在单分子水平。当分析mRNA的亚细胞定位，这是特别重要由于本地化的RNA通常出现在较低的水平。在这里，我们描述的轴突本地化的mRNA使用一种新的超灵敏RNA原位杂交技术检测两个协议。使用荧光免疫组织化学和组织学染料，以验证ATF4 mRNA的招募到轴突在体内成熟小鼠和人的大脑我们结合RNAscope ISH与轴突染液。

的上述过程的简要概述显示在图1中。 利用热诱导揭露ATF4 mRNA的最佳检测颗粒的胆碱能神经轴突当评估mRNA的轴突本地化，它能够识别轴突，并能够可视化低丰度的RNA是至关重要的。这里所描述的RNA原位杂交技术使RNA的检测，在单分子的分辨率。采用这种技术的标准协议建议两个揭露程序的组合，以有效地检测靶RNA：蛋白酶诱导和热诱导...

Watch this Scientific Journal Video about Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization at JoVE.com

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

detection-axonally-localized-mrnas-brain-sections-using-high

Research

JoVE Journal

Neuroscience

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

11.7K 次观看.  Columbia University. RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

视频: Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

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

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...