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。如果背景荧光仍然存在, 漂白持续时间和/或抗体染色步骤需要修改。漂白的持续时间将根据所选择的灯具结构和样品中的自发荧光水平而变化。必须确定每个独特灯具的最佳漂白时间。这可以通过漂白一个不, coverslipped 组织切片安装在 TBS-叠氮化物和测量的内源荧光的幻灯片12小时间隔使用荧光显微镜。在以前的报告中, 我们监测褐漂白的粒子分析和曲线拟合指数衰减函数5, 但为了简单起见, 对各节定期进行目视观察可以同样有效地判断大部分荧光猝灭的持续时间。在我们的情况下, 老年脑组织含有大量的褐色素, 48 h 孵化与800勒克斯灯是足够的。如果与漂白后的背景相比, 特定信号仍然较弱, 考虑在染色过程中增加主次抗体的浓度。同样重要的是要执行一个二级抗体控制, 以确保背景信号不是由非特异性二级抗体结合造成的。注意选择不交叉的抗体, 特别是当染色多靶点时。此外, 确保在染色过程中不会意外地淬火特定的信号。例如, 尼染色 (用于染色神经元) 被 BSA 淬火。在这种情况下, 应该用诸如鱼皮明胶5这样的替代品取代 BSA 组件的阻挡液。
使用白色荧光粉发光二极管的漂白有一定的限制, 由于其发射频谱从 420 nm 到 650 nm。因此, 在紫外波长上没有明显的漂白是可能的。此外, 由于广泛的发射频谱, 漂白团的特定波长需要修改, 如覆盖彩色过滤板上方的 LED 阵列, 以过滤出不良的发射波长。此外, 漂白的组织可能需要比其他方法更长的处理时间。在这种情况下, 由于大脑组织的年龄 (从 89 year-old 个体) 和长醛固定期超过2天, 需要48小时漂白持续时间。虽然没有活动的处理时间是必需的, 你必须考虑到漂白时间并且相应地计划着色会议。更高亮度的 LED 灯或多盏灯的使用可能会显著减少光子通量的简单增加漂白时间。
与现有的方法相比, 我们的技术提供了几个显著的优势。与漂白在荧光显微镜中使用扫描激光器不同, 我们没有观察到使用 LED 漂白对样品造成的光致损伤的迹象。我们设备的另一个主要优点是大大降低了其他技术的成本。我们的设备组装费用约为10美元, 该设备的装配足够灵活, 任何实验室都可以采用他们自己的 photobleacher 版本。相比之下, 使用带有自定义幻灯片持有者和冷却腔的光谱 LED 阵列的其他方法需要多达1000美元来构建8。此外, 商业化学剂是消耗120美元的100张幻灯片的消耗品, 并将可能有害的化合物引入样品中。本研究中用于比较的化学止渴可能含有脂质结合的苏丹黑染料, 这可能会干扰脂蛋白的免疫。
总的来说, 在漂白前处理和免疫中最关键的步骤涉及在免疫期间对该部分的搬运。重要的是, 一旦该节是水化在漂白, 它是不允许干的任何后续步骤。干燥的幻灯片将导致不均匀的抗体分布和创造 uninterpretable 的文物。为了避免失去时间和潜在的宝贵样本, 必须在议定书的几个步骤中加以注意。抗原提取后, 允许组织冷却, 然后转移到 TBS-海卫一性溶液。当样品被加热到高温时, 即使瞬间暴露在空气中, 也会导致组织瞬间干燥。同时确保抗体、阻断或 counterstain 溶液在应用过程中完全覆盖组织。这可以通过确保通过疏水性笔完全勾勒出的组织, 以避免溶液径流, 所有的解决方案都适用于一个水平表面的湿法室。这是特别重要的在夜间孵化 (主要抗体结合)。
在染色前使用白色荧光粉发光二极管漂白的成功应用, 可以促进免疫荧光方法在存档组织中的应用。这是一个多才多艺的和廉价的治疗, 我们期望能够在未来的广泛的标本。虽然我们只将这种方法应用到人脑组织, 但这种技术可以很容易地被纳入到任何染色协议中, 其中的荧光是防止理想的结果, 特别是在 postmitotic 组织, 如心脏或骨骼lipofucin 更有可能积聚的肌肉。

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 border="0" cellpadding="0" cellspacing="0" width="999">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">Trizma Base</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:104px;">T6066</td>
			<td style="width:400px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Choloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hydrochloric Acid</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>1506656</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Azide</td>
			<td>BioShop Canada</td>
			<td>SAZ001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">Citric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>C-2404</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethylenediaminetetraacetic acid&#160;(EDTA)</td>
			<td>BioShop Canada</td>
			<td>EDT001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P-7949</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Hydroxide</td>
			<td>BioShop Canada</td>
			<td>SHY700.1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water bath</td>
			<td>Haake Fisons</td>
			<td>K15</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Slide collector</td>
			<td>FisherScientific</td>
			<td>12-587-17B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Staining Jar</td>
			<td>FisherScientific</td>
			<td>E94</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Orbital Shaker</td>
			<td>Bellco Glass</td>
			<td>&#160;7744-08115</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T7878</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine Serum Albumin</td>
			<td>FisherScientific</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Normal Goat Serum</td>
			<td>Aurion</td>
			<td>905.002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hydrophobic pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z672548-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)</td>
			<td>ThermoFisher</td>
			<td>MN1020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse Secondary Antibody, Texas Red-X</td>
			<td>ThermoFisher</td>
			<td>T862</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mounting medium</td>
			<td>ThermoScientific</td>
			<td>28-600-42</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass soverslip</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software ZEN 2012 Black Edition 11.0</td>
			<td>Zeiss</td>
			<td>LSM710</td>
			<td>Software accompanies the Confocal Microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RGB Profile Tools macro</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 漂白, autofluorescent 斑点的强度类似褐以及一般背景荧光在两个发射波长大大减少了不部分 FTLD t (图 1D)。为了证明漂白的功效, 我们用两种不同的二次抗体对 hyperphosphorylated 头进行染色, 以显示病变的头夹杂物 FTLD。不同形状的 tau 夹杂和使用两个团的标签也使我们能够自信地区分 autofluorescent 的特点, 从预期的目标, 以验证该技术。
在较低放大率的复合图像中, 由 Alexa 488 和德克萨斯州红通道组合而成的头阳性夹杂的形态, 由环状的短细胞过程组成, 称为 "星斑块 "(图 2a-c)。这种形态学特征为 corticobasal 变性 (CBD)12,13, 这与本病例的病理报告一致。在未处理的样品中, 许多结构存在于不像 tau 夹杂的复合图像中, 并且主要在红色通道中显示荧光, 这表明它是自发的, 而不是有意的抗体染色。在整个视图领域 (图 2a) 中, 此通道中也可以看到明显的背景荧光级别。当使用漂白 (PB) 和化学止渴 (图 2b-c) 处理样品时, 这些 autofluorescent 特征被删除, 整体图像显得更加干净。
然后, 我们通过分析每个样本中较小的区域来量化这些样品中的荧光差异。在每个处理条件中都有一个单星斑块供比较 (图 2d-r)。在未经处理的样品, 背景荧光是存在于所有三通道, 但二级抗体荧光为 Alexa 488 和得克萨斯红色抗体是相对地高 (图 2h)。然而, 在未经处理的样品中存在的 autofluorescent 结构在德克萨斯州红色通道中具有荧光强度, 可与为 tau 染色的德克萨斯红二级抗体 (图 2h) 相媲美。如果靶蛋白没有一个明显的, 可预知的形态学, 如 tau 在我们的情况下, 在组织中的自发荧光将呈现图像 uninterpretable。
相比之下, 当漂白前处理之前, 免疫, 背景荧光在 Alexa 488 和得克萨斯州红色通道显着减少相比, 未经处理的样品, 和荧光的 immunostained 头保持不受影响 (图 2i-m)。商业化学止渴在 Alexa 488 通道中猝灭了荧光, 就像漂白一样有效。它还抑制了 DAPI 的背景荧光, 这是不受 48 h 漂白治疗 (图 2m)。然而, 化学止渴也减少了得克萨斯红色次要和 DAPI 信号的强度, 暗示一定程度的适得其反的淬火 (图 2n-r)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56188/56188fig1.jpg"> 
图 1: 漂白预处理在标准免疫荧光工作流程中的应用.A) 从组织获取到成像的标准免疫荧光协议的简化示意图. 主要和二级荧光抗体的应用以卡通为代表。制作了具有代表性的显微镜图像。标尺 = 100 µm. B) 用现成的部件建造的具有代表性的漂白装置 (不显示反射盖)。C) 白二极管阵列的发射谱。观测到450纳米的窄发射峰和 550 nm 的宽峰。D) 漂白对 FTLD 488 (493-570 nm) 和德克萨斯红 (601-635 nm) 发射波长的内源背景荧光的影响。Autofluorescent 斑点类似褐明显减少后, 48 小时漂白。缩放栏 = 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56188/56188fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56188/56188fig2.jpg"> 
图 2:荧光去除对 anti-phosphorylated 头免疫 FTLD 组织的影像质量的影响. a-c) 低放大率、复合免疫荧光图像的代表性视野未经治疗 (a), photobleached 48 h (b) 和化学止渴处理 (c) 样品。颜色代表荧光在以下渠道通过励磁由他们各自的光源: Alexa 488 (绿色): λex = 488 nm (氩激光) λem = 493-570 nm;得克萨斯红色 (红色): λex = 561 nm (DPSS 561 nm 激光器), λem = 601-635 nm;DAPI (蓝色): λex = 405 nm (二极管405激光器), λem = 410-507 nm。缩放条形图 = 100 µm. d-r) 在未经处理的 (d-g), photobleached (i.) 和化学止渴治疗 (n-q) 样品, 具有单独的荧光通道, 合并图像, 和量化荧光信号剖面的斑点区域放大图像。合并通道中的虚线 (g、l、q) 表示生成信号配置文件的行 (h、m、r)。刻度条 = 50 µm Autofluorescent 粒子 (af)immunolabeled 头荧光 (tau) 和核信号 (校正) 被表明。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56188/56188fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

科研

生物化学

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

体外荧光显微镜基于动感检测的肌素特异性适应

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...