A new method for visualization of endothelial cells and extravascular leakage in adult mouse brain using fluorescein isothiocyanate
A new method for visualization of endothelial cells and extravascular leakage in adult mouse brain using fluorescein isothiocyanate
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DOI：
10.1016/j.jneumeth.2011.08.002
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发表时间：
2011-10-30
影响因子：
3
通讯作者：
Morita, Shoko
中科院分区：
文献类型：
--
作者：
Miyata, Seiji;Morita, Shoko
我们描述了一种新的方法，血管和内皮细胞的可视化和评估血管外渗漏在成年小鼠大脑中使用异硫氰酸荧光素（FITC），或活性荧光染料。FITC是在碱性pH值下与胺基共价反应的荧光素衍生物。在该方法中，当小鼠接受心内灌注含FITC的生理盐水（pH 7.0），然后用多聚甲醛（PFA）固定剂（pH 8.0）时，在整个脑和脊髓的血管系统中观察到FITC的强荧光。在灌注pH 7.0的含FITC的盐水后，当动物用pH 7.0的PFA固定剂固定时，FITC的荧光微弱。在灌注含FITC的生理盐水之前，用PFA固定剂固定小鼠，未检测到FITC的荧光。用内皮细胞标记物CD 31或周细胞标记物结蛋白进行免疫组化双标记，结果显示FITC聚集在内皮细胞的细胞核上，而周细胞的细胞核上没有。FITC的血管外渗漏在最后区或缺乏血脑屏障的脑室周围器官的脑区中突出。此外，在冷冻损伤的大脑皮层损伤部位检测到强烈的血管外FITC渗漏。因此。FITC法是研究成年啮齿类动物脑血管和内皮细胞构筑及血管外渗漏的有效方法。而且。FITC与细胞组分共价结合，因此可以进行双标记免疫组织化学和长期储存。(C)2011 Elsevier B.V.保留所有权利。
We described a new method for the visualization of vasculature and endothelial cells and the assessment of extravascular leakage in adult mouse brain by using fluorescein isothiocyanate (FITC), or a reactive fluorescent dye. FITC is the fluorescein derivative that reacts covalently with amine groups at alkaline pH. In this method, strong fluorescence of FITC was seen at vasculature throughout the brain and spinal cord, when mice received intracardiac perfusion with FITC-containing saline at pH 7.0 followed by paraformaldehyde (PFA) fixative at pH 8.0. The fluorescence of FITC was faint when animals were fixed with PFA fixative at pH 7.0 after the perfusion of FITC-containing saline at pH 7.0. The fluorescence of FITC was not detected when mice was fixed with PFA fixative before the perfusion of FITC-containing saline. Double labeling immunohistochemistry using an endothelial cell marker CD31 or a pericyte marker desmin revealed that FITC was accumulated at nuclei of endothelial cells but riot at those of pericytes. Extravascular leakage of FITC was prominent in the area postrema or a brain region of the circumventricular organs that lacks the blood-brain barrier. Moreover, strong extravascular leakage of FITC was detected at damaged sites of the cerebral cortex with cryoinjury. Thus. FITC method is useful technique for examining the architecture of brain vasculature and endothelial cells and the assessment of extravascular leakage in adult rodents. Moreover. FITC binds covalently to cellular components, so that makes it possible to perform double labeling immunohistochemistry and long-term storage of the preparation. (C) 2011 Elsevier B.V. All rights reserved.