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 C. Sealfon

doi:10.1117/12.661780

27 March 2006 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 C. Sealfon

Author Affiliations +

Proceedings Volume 6096, Colloidal Quantum Dots for Biomedical Applications; 609614 (2006) https://doi.org/10.1117/12.661780
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

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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PokMan Chan, Gang Lin, Tony Yuen, Badrinath Roysam, and Stuart C. Sealfon "Development of multiplex immunohistochemistry and in situ hybridization using colloidal quantum dots for semi-automated neuronal expression mapping in brain", Proc. SPIE 6096, Colloidal Quantum Dots for Biomedical Applications, 609614 (27 March 2006); https://doi.org/10.1117/12.661780

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KEYWORDS

Brain

Neurons

Proteins

Signal detection

Tissues

Image analysis

Image segmentation

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