Name: simple elimination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy
Uploaded: 2017-09-03T13:00:00
Description: Watch this Scientific Journal Video about Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy at JoVE.com

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) ...

<ol>
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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 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 ?...

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。

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