Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease
Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease
复制标题
DOI：
10.1177/002215540305100910
复制
发表时间：
2003-09-01
影响因子：
3.2
通讯作者：
Mizushima, S
中科院分区：
文献类型：
--
作者：
Uchihara, T;Nakamura, A;Mizushima, S
我们用两种兔多克隆抗体和一种小鼠单克隆抗体建立了三重标记方法，并检查了带有棉絮斑块（CWP）的尸检脑组织。将其中一种多克隆抗体稀释（抗Abeta 42或抗Abeta 40/1：30，000或抗von Willebrand因子/1：1000），使其仅在用催化报告沉积（CARD）方法扩增后才可能可视化。另一种多克隆抗体（抗-Abeta 40或抗-Abeta 42/1：1000）用与抗兔抗体偶联的荧光染料显色，由于后者的灵敏度较低，该抗体特异性显色。将单克隆抗体AT 8叠加以产生三重免疫荧光标记。重建间隔为0.3 μ m的连续光学切片，以允许对这三个表位进行三维（313）观察。A β 40定位于核心样结构，主要在I-III层，有时与血管壁接触，两者均无神经炎反应。CWP，目前生病层I-VI，用抗A β 42标记，并伴有神经炎反应。这些差异表明，即使在相同的显微镜视野中，A β沉积的机制及其与神经炎反应或血管的关系也因病变而异。
We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Abeta42 or anti-Abeta40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Abeta40 or anti-Abeta42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 mum were reconstructed to allow three-dimensional (313) observation of these three epitopes. Abeta40 was localized to core-like structures, mainly in layers I-III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present ill layers I-VI, were labeled with anti-Abeta42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Abeta deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.