Padala Sanjana, Setti Sharay, Raymick James, Hanig Joseph, Sarkar Sumit
Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR72079, USA.
Office of Testing & Research, Center for Drug Evaluation Research/FDA, Silver Spring, MD, USA.
Curr Alzheimer Res. 2024;21(1):69-80. doi: 10.2174/0115672050295561240327055835.
A plethora of studies has shown the utility of several chemical dyes due to their affinity to bind Aβ to enable visualization of plaques under light or fluorescence microscope, and some of them showed affinity to bind neurofibrillary tangles (NFT) as well. However, only a few of them have the propensity to bind both senile plaques (SP) and NFT simultaneously.
In our current study, we aimed to modify the K114 dye and the staining procedure to substantially improve the staining of amyloid plaques in both human and rodent brains and neurofibrillary tangles in the human brain.
We modified the K114 solution and the staining procedure using Sudan Black as a modifier. Additionally, to evaluate the target of the modified K114, we performed double labeling of K114 and increased Aβ against three different epitopes. We used 5 different antibodies to detect phosphorylated tau to understand the specific targets that modified K114 binds.
Dual labeling using hyperphosphorylated antibodies against AT8, pTau, and TNT1 revealed that more than 80% hyperphosphorylated tau colocalized with tangles that were positive for modified K114, whereas more than 70% of the hyperphosphorylated tau colocalized with modified K114. On the other hand, more than 80% of the plaques that were stained with Aβ MOAB-2 were colocalized with modified K114.
Our modified method can label amyloid plaques within 5 min in the rat brain and within 20 min in the human brain. Our results indicated that modified K114 could be used as a valuable tool for detecting amyloid plaques and tangles with high contrast and resolution relative to other conventional fluorescence markers.
大量研究表明，几种化学染料具有实用性，因为它们能够与β-淀粉样蛋白（Aβ）结合，从而在光学显微镜或荧光显微镜下使斑块可视化，其中一些染料还显示出与神经原纤维缠结（NFT）结合的亲和力。然而，其中只有少数染料有同时与老年斑（SP）和NFT结合的倾向。
在我们当前的研究中，我们旨在对K114染料和染色程序进行改进，以大幅提高在人类和啮齿动物大脑中淀粉样斑块以及人类大脑中神经原纤维缠结的染色效果。
我们使用苏丹黑作为改性剂对K114溶液和染色程序进行了改进。此外，为了评估改性K114的靶点，我们针对三种不同表位对K114和增加的Aβ进行了双重标记。我们使用5种不同的抗体来检测磷酸化tau，以了解改性K114所结合的特定靶点。
使用针对AT8、pTau和TNT1的高磷酸化抗体进行双重标记显示，超过80%的高磷酸化tau与改性K114阳性的缠结共定位，而超过70%的高磷酸化tau与改性K114共定位。另一方面，用Aβ MOAB-2染色的斑块中超过80%与改性K114共定位。
我们改进后的方法可在大鼠大脑中5分钟内、人类大脑中20分钟内标记淀粉样斑块。我们的结果表明，相对于其他传统荧光标记物，改性K114可作为一种有价值的工具，用于以高对比度和分辨率检测淀粉样斑块和缠结。
