Evaluation and Characterization of Modified K114 Method to Localize Plaques in Rodent and Plaques and Tangles in Human Brain Tissue


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Abstract

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.

About the authors

Sanjana Padala

Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration

Email: info@benthamscience.net

Sharay Setti

Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration

Email: info@benthamscience.net

James Raymick

Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration

Email: info@benthamscience.net

Joseph Hanig

Office of Testing & Research, Center for Drug Evaluation Research/FDA

Email: info@benthamscience.net

Sumit Sarkar

Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration

Author for correspondence.
Email: info@benthamscience.net

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