Background: 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. Objective: 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 Methods: 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. method: We have modified the K114 solution and the staining procedure using Sudan Black as modifier. Additionally, to evaluate the target of the modified K114, we performed double labeling of K114, and Aβ raised against three different epitopes. We used 5 different antibodies to detect phosphorylated Tau to understand the potential binding targets. Results: 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. result: We have found more than 80% hyperphosphorylated Tau against AT8, PTau and TNT1 colocalized with K114 labeled tangles, 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 amyloid beta MOAB-2 were colocalized with modified K114. Conclusion: 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.

中文翻译：

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改进的 K114 方法定位啮齿动物​​斑块以及人脑组织斑块和缠结的评估和表征

背景：大量研究表明，几种化学染料具有与 Aβ 结合的亲和力，可在光学或荧光显微镜下观察斑块，其中一些化学染料还具有与神经原纤维缠结 (NFT) 结合的亲和力。然而，其中只有少数具有同时结合老年斑（SP）和 NFT 的倾向。目的：在我们当前的研究中，我们的目的是修改 K114 染料和染色程序，以显着改善人类和啮齿动物大脑中淀粉样斑块以及人脑中神经原纤维缠结的染色方法：我们修改了 K114 溶液和染色程序使用苏丹黑作为改性剂。此外，为了评估修饰后的 K114 的靶点，我们对 K114 进行了双重标记，并增加了针对三个不同表位的 Aβ。我们使用 5 种不同的抗体来检测磷酸化 tau 蛋白，以了解修饰后的 K114 结合的特定靶点。方法：我们使用苏丹黑作为改性剂改进了K114溶液和染色程序。此外，为了评估修饰的 K114 的靶标，我们对 K114 进行了双重标记，并针对三个不同的表位引发了 Aβ。我们使用 5 种不同的抗体来检测磷酸化 Tau，以了解潜在的结合靶点。结果：使用针对 AT8、pTau 和 TNT1 的过度磷酸化抗体进行双重标记显示，超过 80% 的过度磷酸化 tau 与修饰 K114 呈阳性的缠结共定位，而超过 70% 的过度磷酸化 tau 与修饰 K114 共定位。另一方面，超过 80% 的 Aβ MOAB-2 染色斑块与修饰的 K114 共定位。结果：我们发现超过 80% 的针对 AT8、PTau 和 TNT1 的过度磷酸化 Tau 与 K114 标记的缠结共定位，而超过 70% 的过度磷酸化 Tau 与修饰的 K114 共定位。另一方面，超过 80% 的淀粉样蛋白 β MOAB-2 染色斑块与修饰的 K114 共定位。结论：我们的改进方法可以在 5 分钟内标记大鼠大脑中的淀粉样斑块，在 20 分钟内标记人大脑中的淀粉样斑块。我们的结果表明，相对于其他传统荧光标记物，修饰的 K114 可用作检测淀粉样蛋白斑和缠结的有价值的工具，具有高对比度和分辨率。