Mr. Stuart C. Sealfon Profile

Stuart C. Sealfon

at Mount Sinai School of Medicine

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Proceedings Article | 27 March 2006 Paper

Development of multiplex immunohistochemistry and in situ hybridization using colloidal quantum dots for semi-automated neuronal expression mapping in brain

PokMan Chan, Gang Lin, Tony Yuen, Badrinath Roysam, Stuart Sealfon

Proceedings Volume 6096, 609614 (2006) https://doi.org/10.1117/12.661780

KEYWORDS: Brain, Neurons, Proteins, Tissues, Signal detection, Image analysis, Image segmentation, Target detection, Quantum dots, Brain mapping

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The number of different subtypes of neurons, which form the basic component of the mammalian brain, has not been determined. Histological study is typically limited to the simultaneous detection of very few markers, in part because of the spectral overlap and quenching properties of organic fluorophores. The photostability and narrow emission spectra of non-organic colloidal quantum-dot fluorophores (QDs) make them desirable candidates for multiplex immunohistochemistry (IHC) and for fluorescent in situ hybridization (FISH). IHC is used to study specific protein epitopes and FISH to study the expression of specific mRNA transcripts. In order to investigate the patterns of coexpression of multiple specific protein and nucleic acid targets within cells in complex tissues, such as brain, we have developed protocols for the multiplex use of different QDs and organic fluorophores for combined IHC and FISH. We developed a method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions and validated this technique in mouse brainstem sections. The reproducible histological results obtained with this protocol allow the use of high throughput computer image analysis to quantify the cellular and subcellular spatial pattern of expression of all markers studied. This approach is being utilized to generate a multiplex co-expression map of neuronal subtypes in mouse brain regions.

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