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제120회 대한화학회 학술발표회, 총회 및 기기전시회 안내 Iterative antibody reprobing for optical imaging in nano-scale

2017년 8월 28일 12시 58분 01초
ACBI-7 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
목 14시 : 10분
Analytical Chemistry-Life Chemistry - [Analytical Chemistry Division - JASIS (Japan) - Life Chemistry Division Joint Symposium] Super-Resolution Optical Microscopy and Single-Cell Analysis
저자 및
Sunghoe Chang
Physiology and Biomedical Sciences, Seoul National University, Korea
Despite its enormous usefulness, the current method for simultaneous immunostaining of multiple protein targets requires the primary antibody to be raised in different species, and that the secondary antibodies recognize one of the species exclusively, which put a limit on the number of concurrent visualization of multiple targets. Several SDS-based stripping methods for antibody reprobing have been developed but they suffer from the unavoidable distortion of cellular microstructures and the residual immunoreactivity due to an incomplete removal of antibodies. Here, we develop a new antibody stripping method for optical imaging in nano-scale. We found new method completely striped primary and secondary antibodies off without damaging any cellular structures, allowing us to visualize distinct multiple target proteins sequentially using corresponding multiple primary antibodies regardless of the species, even from the same species. We applied it to the super-resolution stochastic optical reconstruction microscope (STORM). We found iterative STORM imaging unambiguously registered the same locations of undeformed cellular structures with sub-diffraction limit resolution. Furthermore, we applied it for temporal multiplexing with different primary antibodies against distinct target proteins followed by the same Alexa-647 conjugated secondary antibody, and found that iterative STORM imaging with temporal multiplexing clearly visualized five distinct target proteins with subdiffraction resolution without any overlap or mis-localization due to residual antibodies. In summary, we have demonstrated that our method is capable of visualizing multiple biological structures regardless of the species from which the primary antibodies were raised. By combining with temporal multiplexing, this is highly scalable.