这项研究得到了加拿大神经和老龄协会 (CCNA)、加拿大卫生研究所 (研究院)、加拿大 als 协会 (加拿大) 和加拿大阿尔茨海默氏症协会 (ASC) 的全部或部分支持。作者想感谢来自海洋脑组织的苏丹 Darvesh 和安德鲁 Reid 提供 FTLD 的脑组织, 米兰 Ganguly 从时空目标和放大辐射响应 (STTARR) 计划及其附属用于组织植入和切片服务的供资机构, 以及提供显微仪器的先进光学显微镜设备 (AOMF)。凯文. 哈德利对手稿的评论非常感谢。

注意: 所介绍的工作是按照公认的国际标准执行的, 包括国际协调会议 (理事会)、国际医学组织理事会和《赫尔辛基宣言》的原则。人体组织的使用是由大学卫生网络研究伦理委员会批准的。人脑标本被收集为海洋脑组织库的一部分。在收集时, 所有患者均获得了知情同意. 
 1. 漂白设备和解决方案的构造 <ol> <li> 准备库存解决方案。
	<ol> <li> 准备 1 L 1x 库存三缓冲盐水 (1x tb) 溶液 (150 毫米氯化钠, 50 毫米三氯, ph 7.4) 溶解8.77 克氯化钠和6.06 克的三基在800毫升的 ddH 2 O, 并调整 ph 值为7.4 使用 HCl. 把音量调到1升和高压釜。李 > <li> 准备 10% (200x 股) 叠氮化钠, 在10毫升 ddH 2 O (10%, 200x 库存) 溶解1克叠氮化钠. </li>
	</ol> </li> <li> 对于1-3 标准大小的幻灯片, 使用单个 100 mm x 100 mm 透明的方形培养皿作为滑动腔。对于一个单一的滑动室, 增加0.25 毫升的200x 钠叠氮化物到50毫升的 1x tbs, 使0.05% 叠氮化-tbs 解决方案。堆叠2-3 幻灯片室垂直处理其他幻灯片。为每个腔室准备另外50毫升的叠氮化-TBS 溶液. </li>
	<li> 创建一个脚手架, 以提高幻灯片室 (s), 使灯头可以容纳在下面。
	<ol> <li> 对于 100 mm x 100 mm 的滑动腔, 在底部和两侧切开一个 100 mm x 100 mm x 30 mm 的塑料食品容器并倒置容器。确保侧面开口足够大, 以适合 LED 光源, 并确保底部开口足够大, 这样光源的光线到达样品室时不会产生障碍物. </li>
		<li> 将电子胶带应用于脚手架, 以增加脚手架和样品室/台式之间的握杆。使用任何替代材料建造脚手架, 只要它安全地提升幻灯片室, 而不妨碍光线到达样品. </li>
	</ol> </li> <li> 从台灯上卸下任何扩散或不透明的塑料, 可能会阻碍 led 光源直接到达样品 (如果可能), 并将 led 阵列向上定向。将支架和滑动腔置于 LED 阵列上方。使用灵活的颈部灯, 便于操作. </li>
	<li> 通过在一个大得足以盖住滑动室和带有铝箔的支架的盒子内衬, 为仪器构造反射式圆顶罩。使用1毫升吸管尖端箱为一个单一的房间或150毫米 x 150 毫米 x 150 毫米纸板箱为多个, 垂直堆积商会. </li>
</ol> 2。组织切片的漂白前处理 注意: 组织切片的制备可能因组织和固定的来源以及所使用的嵌入方法而异。在这里, 脑组织 (前额回) 从 FTLD 的情况下固定为〜2天的福尔马林, 运行通过蔗糖梯度, 嵌入 OCT, 并削减到10和 #181; m 厚部分使用低温. 
<ol> <li> 在4和 #186; C 冷室、冷柜或冰箱, 将灯置于支架下, 并将样品箱放在脚手架上。将50毫升叠氮化-TBS 溶液倒入样品室. </li>
	<li> 将组织切片安装在标准玻璃显微镜下, 用干净的镊子将其放入含有叠氮化物-TBS 的滑动腔中。对于多张幻灯片, 请确保将幻灯片放在单个图层的会议厅中. </li>
	<li> 用反光罩盖住设备, 打开 LED 灯, 并在4和 #186 上孵育48小时; C. </li>
</ol> 3。免疫荧光 <ol> <li> 使用 DAPI counterstain 和 Alexa 488-和德克萨斯州红共轭二级抗体染色磷酸化 tau 组织, 首先准备用于抗原检索、性、阻断和初级抗体结合。
	<ol> <li> 准备500毫升的抗原检索缓冲 (10 毫米柠檬酸, 2 毫米乙酸酸, 0.05% 吐温 20; ph 6.2) 溶解0.92 克柠檬酸和0.37 克乙酸酸 (EDTA) 在500毫升 ddH 2 o. 调整 pH 值到6.2 与氢氧化钠和增加0.25 毫升吐温 20. </li>
		<li> 准备500毫升的0.025% 海卫 X-100 在 tbs 解决方案 (tbs-海卫一) 通过增加0.125 毫升的海卫 X-100 到500毫升 1x tbs. </li>
		<li> 将1% 牛血清白蛋白 (bsa) 溶液在 tbs (bsa-tbs 缓冲液) 中溶解 0.1 g bsa, 在10毫升 tbs. </li>
		<li> 通过将正常山羊血清的0.2 毫升添加到1.8 毫升的 1% BSA/TBS, 准备一个阻断溶液. </li>
		<li> 对于每张幻灯片, 准备150和 #181; 移1.5 和 #181 的初级抗体溶液 (1:100 稀释); anti-phospho-PHF 头 pSer202 + Thr205 (AT8) 抗体进入148.5 和 #181; l 1% BSA-TBS 缓冲, 并留在冰上. </li>
	</ol> </li> <li> 执行抗原检索, 将 photobleached 幻灯片垂直淹没在包含25毫升抗原检索缓冲区的幻灯片收集器中。使用磁带和/或字符串保护收集器, 使收集器不落入水浴。在90和 #186 的水浴中加热收集器; C 为30分钟, 使收集器在移除幻灯片前将温度冷却到室温30分钟。不要立即删除幻灯片, 因为它会导致部分干涸. </li>
	<li> 将从抗原检索收集器的幻灯片转到一个充满了30毫升 TBS-海卫一的染色罐子, 并在一个温和晃动的轨道振动筛上清洗5分钟的切片。用新鲜的 TBS-海卫一重复一次洗涤。用不起毛的纸巾将多余的缓冲器吸走, 用疏水的钢笔勾勒出组织的轮廓。当心不要让滑梯变干。
	<ol> <li> 对于每张幻灯片, 用移200和 #181 阻止该组织, 将溶液阻塞在组织上, 并将滑块放置在湿化室中。在装有湿纸巾的吸管尖盒内放置一个滑动架, 来建造房间。在室温下孵育 2 h 级表面。确保阻塞解决方案完全覆盖了组织. </li>
	</ol> </li> <li> 通过吸入和吸管100-150 和 #181 将阻塞解决方案移到组织的主抗体溶液中。确保有足够数量的抗体存在, 该部分是在一个水平表面上, 以避免汇集抗体解决方案的一侧。在4和 #186 上孵育, 在湿润的室内过夜. </li>
	<li> 制备二次抗体混合物和 DAPI 核 counterstain。
	<ol> <li> 对于每张幻灯片, 准备150和 #181; 二级抗体混合物 (1:100 稀释), 加入1.5 和 #181; 山羊 anti-mouse 488 和1.5 和 #181; l 山羊 anti-mouse 得克萨斯红色到147和 #181; l BSA-TBS 和离开冰. </li>
		<li> 准备0.1 和 #181; 通过串联稀释 DAPI counterstain。将库存解决方案彻底混合, 稀释1和 #181; L 库存5毫克/毫升 DAPI 在999的 TBS, 使1毫升5和 #181; g/毫升解决方案。每张幻灯片, 稀释3和 #181;5和 #181 的 l; 147 和 #181 的 g/ml 解决方案; TBS 的最终浓度为0.1 和 #181; g/毫升. 
		注意: DAPI 是已知的诱变, 应该小心处理. </li>
	</ol> </li> <li> 通过吸入移除主抗体。将幻灯片浸入装有30毫升 TBS 的玻璃染色罐中, 并在轨道振动筛上轻轻搅拌5分钟。用新鲜的 TBS-海卫一重复清洗步骤。将过量的 TBS-海卫一和吸管100至150和 #181; 二次抗体混合物的每张幻灯片。
	<ol> <li> 确保组织完全由抗体混合物覆盖。在室温下, 在黑暗的湿润室内孵育2小时. </li>
	</ol> </li> <li> 通过吸入去除二次抗体混合物, 并将滑块转移到含有30毫升 TBS (无海卫一) 的玻璃染色罐中。在轨道振动筛上轻轻搅拌5分钟。用新鲜的 TBS 重复洗涤步骤. 应用100至150和 #181; L 0.1 和 #181; DAPI counterstain 每张幻灯片, 在黑暗中室温下孵育10分钟. </li>
	<li> 将幻灯片转到含有 tbs 的玻璃着色罐中, 用温和的混合方法洗涤3次, 每次洗涤时使用新鲜的 tbs。吸走多余的缓冲器. </li>
	<li> 在组织中应用3滴水性安装介质。使用镊子, 轻轻地降低一个干净的玻璃片到组织, 从一个边缘开始, 并慢慢降低其他边缘, 以避免捕捉气泡。注意不要把片, 如果立即成像。否则, 在4和 #186 储存前, 允许安装介质干燥; C 在黑暗中. </li>
</ol> 4。荧光显微镜 <ol> <li> 打开萤光灯、显微镜和电脑, 让灯预热15分钟, 将染色组织切片放置在荧光显微镜阶段。使用明亮的字段图像在10x 放大时定位组织. </li>
	<li> 应用一滴 ddH 2 O 到片表面, 并使用20x 水浸泡物镜 (NA = 1.0)。选择4行平均平面扫描设置。将针孔大小设置为1通风单元, 该单位提供3微米的光学切片。选择激光激发和发射波长为每个荧光在不同的轨道为最佳的信号. 
	注: Alexa 488: & #955; ex = 488 nm (氩激光) 和 #955; em = 493-570 nm;得克萨斯红色: 和 #955; ex = 561 nm (DPSS 561 nm 激光器), 和 #955; em = 601-635 nm;DAPI: 和 #955; ex = 405 nm (二极管405激光器), 和 #955; em = 410-507 nm。
	<ol> <li> 调整激光功率和增益设置, 以优化每个轨道的信号强度。收集复合图像并保存。使用相同的激光设置来比较不同的幻灯片中的荧光强度. </li>
	</ol> </li> <li> 对于每个通道中的荧光强度可视化, 请安装 ImageJ 的 RGB 配置文件工具宏 10 。将该宏从网页保存为文本文件 (https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt)。从 ImageJ 菜单中选择插件--#62; 宏和 #62; 安装;选择要安装 RGB 配置文件工具的文本文件。
	<ol> <li> 在 ImageJ 中打开共焦图像文件, 并通过执行以下操作将复合图像从3堆栈转换为 RGB: 图像-#62; 彩色 #62; 通道工具。选择和 #34; 复合和 #34; 从下拉菜单中检查所有三通道。然后, 选择图像和 #62; 类型和 #62; RGB 颜色. </li>
		<li> 和 #160; 选择 RGB 配置文件工具图标, 并在要分析的图像中绘制一条线。将强度数据保存为绘图的电子表格. </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有什么可透露的。

本手稿中所描述的组织的漂白预处理可以有效地消除使用现成组件的荧光。该协议描述了在福尔马林固定的人脑组织中磷酸化 tau 集料的免疫荧光成像, 使用继发性抗体共轭于 Alexa 488 和得克萨斯州红, DAPI 作为核 counterstain。为了将该方法应用于其他组织, 我们建议对样品进行48漂白前处理, 作为出发点。漂白后, 执行标准的免疫荧光染色协议的特点, 使用抗体稀释根据制造商的建议。如果背景荧光仍?...

荧光显微镜使用的抗体靶向特定的蛋白质是常规用于可视化蛋白的兴趣在细胞培养和组织。一个主要的并发症, 以获取清晰和明确的图像在免疫荧光是自发荧光, 这可以导致内哺乳动物组织的年龄色素褐和蛋白质, 如弹性蛋白和胶原蛋白1, 2。其他来源的自发荧光可以通过样品制备步骤, 如醛固定3。褐颗粒, 主要由氧化修饰蛋白和脂质降解残留物组成, 在运行细胞中积累, 年龄增加了2。这会导致成像 postmitotic 组织如大脑和心脏或骨骼肌肉的困难, 因为褐的荧光发射谱是广泛的和可变的, 往往与共同荧光的发射波长, 用于标记4。这些因素使人类脑组织的成像从晚发性神经退行性疾病, 如颞肺叶变性 (FTLD), 特别是挑战。
为了减少自发荧光, 我们设计了一种技术, 我们使用一盏家用台灯5, 用白色发光二极管 (LED) 阵列对滑动安装的组织切片进行照射。这种简单的技术提供了一种替代技术, 使用化学剂, 如丘索4在醋酸铵, 或商业可用的淬火染料, 如苏丹黑 B 和铬黑 T6。它还具有显著的成本节约多光谱 LED 灯漂白技术, 避免并发症和文物产生的数字自体荧光去除方法, 如光谱 un-mixing7,8。白色荧光粉发光二极管具有广泛的发射光谱、高亮度和低制造成本, 使其成为漂白各种团59的现成组件的理想选择。
在本协议中, 我们演示了使用可拆卸元件的漂白装置的构造, 并将漂白应用于含 tau 阳性包裹体 (FTLD-T) 的 FTLD 组织, 并使用特定于磷酸化 tau 的抗体。我们演示了漂白对使用两个常用团的成像荧光标记抗体的影响: Alexa 488 和德州红。漂白与未经处理的部分或那些与商业化学止渴的治疗效果的量化和比较。这种漂白预处理可以纳入任何标准的免疫荧光染色协议, 以消除在生物样品中的荧光。

<table><tbody><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T6066</td><td></td></tr><tr><td>Sodium Choloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Caledon Laboratory Chemicals</td><td>1506656</td><td></td></tr><tr><td>Sodium Azide</td><td>BioShop Canada</td><td>SAZ001</td><td></td></tr><tr><td>100 mm x 100 mm x 20 mm Pitri dish</td><td>Sarstedt</td><td>82.9923.422</td><td>All components of photobleacher can be substituted based on availability</td></tr><tr><td>6 W LED Dimmable Desk Lamp</td><td>DBPower</td><td>DS501</td><td>All components of photobleacher can be substituted based on availability</td></tr><tr><td>Citric Acid</td><td>Sigma-Aldrich</td><td>C-2404</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;(EDTA)</td><td>BioShop Canada</td><td>EDT001</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P-7949</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>BioShop Canada</td><td>SHY700.1</td><td></td></tr><tr><td>Water bath</td><td>Haake Fisons</td><td>K15</td><td></td></tr><tr><td>Slide collector</td><td>FisherScientific</td><td>12-587-17B</td><td></td></tr><tr><td>Staining Jar</td><td>FisherScientific</td><td>E94</td><td></td></tr><tr><td>Orbital Shaker</td><td>Bellco Glass</td><td>&amp;nbsp;7744-08115</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T7878</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>FisherScientific</td><td>BP1600-1</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Aurion</td><td>905.002</td><td></td></tr><tr><td>Hydrophobic pen</td><td>Sigma-Aldrich</td><td>Z672548-1EA</td><td></td></tr><tr><td>Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)</td><td>ThermoFisher</td><td>MN1020</td><td></td></tr><tr><td>Goat anti-Mouse Secondary Antibody, Texas Red-X</td><td>ThermoFisher</td><td>T862</td><td></td></tr><tr><td>Goat anti-Mouse Secondary Antibody, Alexa Fluor 488</td><td>ThermoFisher</td><td>A-11029</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Mounting medium</td><td>ThermoScientific</td><td>28-600-42</td><td></td></tr><tr><td>Glass soverslip</td><td></td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM710</td><td></td></tr><tr><td>Imaging software ZEN 2012 Black Edition 11.0</td><td>Zeiss</td><td>LSM710</td><td>Software accompanies the Confocal Microscope</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>RGB Profile Tools macro</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt</td></tr><tr><td>Commercial chemical quencher</td><td>Biotum</td><td>23007</td><td></td></tr></tbody></table>

simple elimination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy

免疫荧光法是一种常见的方法, 用于可视化亚细胞室和确定特定的蛋白质在组织样本的定位。高品质免疫荧光图像获取的一个很大的障碍是由衰老色素引起的组织内源性自发荧光, 如褐或普通的样品制备过程, 如醛固定。该协议描述了如何通过使用荧光探针处理前的白光发光二极管 (LED) 阵列, 大大降低背景荧光的漂白。白色荧光粉发光二极管的 broad-spectrum 排放允许在一系列的发射峰上漂白荧光。漂白设备可以从现成的组件, 以非常低的成本, 并提供了一个可供选择的商业可用的化学剂。漂白前处理的组织和常规免疫荧光染色产生的图像没有背景自发荧光。与已建立的化学剂相比, 降低探针和背景信号, 漂白处理对探针荧光强度没有影响, 有效降低了背景和褐荧光。虽然漂白需要更多的时间进行预处理, 但更高强度的 LED 阵列可以用来减少漂白时间。这种简单的方法可能适用于各种组织, 特别是 postmitotic 组织, 积累褐, 如大脑和心脏或骨骼肌肉。

在抗原检索和免疫 (图 1A) 之前, 漂白预处理步骤可以添加到标准免疫荧光协议中。漂白设备的组装也可以使用各种廉价的现成组件 (图 1B) 进行。白色荧光粉发光二极管的发射光谱涵盖了宽范围的波长, 使它们适合广泛的漂白, 同意以前的报告 (图 1C)5,11。在 48 h ?...

Watch this Scientific Journal Video about Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy at JoVE.com

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

荧光显微镜下福尔马林固定人脑组织背景荧光的简单消除

生物样品的背景自发荧光常常使荧光成像技术复杂化, 特别是在老年人类 postmitotic 组织中。本协议描述了如何有效地去除这些样品的自体荧光, 利用商业上可用的发光二极管光源在免疫之前 photobleach 样品。

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue ...

simple-elimination-background-fluorescence-formalin-fixed-human-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

17.1K 次观看.  University of Toronto. 生物样品的背景自发荧光常常使荧光成像技术复杂化, 特别是在老年人类 postmitotic 组织中。本协议描述了如何有效地去除这些样品的自体荧光, 利用商业上可用的发光二极管光源在免疫之前 photobleach 样品。

视频: Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

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 ...

科研

神经科学

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

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

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 ...

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 ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

冷冻组织部分多光谱荧光成像的快速方法

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

免疫与感染

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

小鼠大脑的自由漂浮免疫染色

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

使用 SHORT 和 LSFM 对人脑进行高分辨率 3D 重建

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

作者聚焦：推进 3D 细胞结构分析 - 人脑的快速体积重建 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

荧光显微镜下福尔马林固定人脑组织背景荧光的简单消除

摘要

探索更多视频

此视频中的章节

一个快速高分辨率的荧光成像的方法在半厚的脑片

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

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

冷冻组织部分多光谱荧光成像的快速方法

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

小鼠大脑的自由漂浮免疫染色

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

荧光显微镜下福尔马林固定人脑组织背景荧光的简单消除

摘要

探索更多视频

此视频中的章节

一个快速高分辨率的荧光成像的方法在半厚的脑片

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

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

冷冻组织部分多光谱荧光成像的快速方法

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

小鼠大脑的自由漂浮免疫染色

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究