Division of Neurotoxicology, National Center for Toxicological Research/US FDA, HFT-132, Jefferson, AR, 72079, USA.
Office of Testing & Research, Center for Drug Evaluation Research/FDA, Silver Spring, MD, USA.
Metab Brain Dis. 2020 Dec;35(8):1371-1383. doi: 10.1007/s11011-020-00608-0. Epub 2020 Aug 27.
Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aβ) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aβ as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aβ. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.
尽管有多种组织化学示踪剂可用于标记大脑中的斑块和缠结，以评估阿尔茨海默病（AD）的神经病理学，但它们中很少有具有多功能性且与免疫组织化学程序兼容的。刚果红（CR）是一种各向异性的有机染料，用于标记大脑中的淀粉样β（Aβ）斑块。不幸的是，由于其在先前使用明场显微镜的研究中分辨率和亮度低，其使用未得到充分重视。在这里，我们改进了以前的方法，用于在人类大脑和 AD 的转基因啮齿动物模型中荧光显微镜可视化来定位斑块和缠结。CR 显示的斑块染色亲和力与 Thioflavin S 显示的纤维状模式标记进行了比较。这项研究总结了优化方案，其中对各种参数进行了微调。为了确定 CR 潜在结合的靶标，我们已经用针对 Aβ和磷酸化 Tau 的不同抗体进行了双重标记。CR 显示的斑块染色亲和力与针对 Aβ不同表位的抗体的弥散模式标记相关联的亲和力进行了比较。CR 标记在人类大脑中揭示，CP13、TNT2 或 TOC1 均不能结合所有神经原纤维缠结。此外，我们还评估了与 AT8、AT180 和 PHF1 的双重标记。有趣的是，PHF-1 与 NFT 显示 40％的共定位，而 AT8 显示 15％的共定位。因此，CR 是一种更好的标记物，可用于检测人类和啮齿动物大脑中的 AD 病理学，其荧光强度相对其他传统荧光标记物更高。
