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Nucleic Acids Research 2005 33(18):e161; doi:10.1093/nar/gni162
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Published online 13 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization

PokMan Chan, Tony Yuen, Frederique Ruf, Javier Gonzalez-Maeso and Stuart C. Sealfon*

Department of Neurology, Mount Sinai School of Medicine One Gustave L. Levy Place, NY 10029, USA

*To whom correspondence should be addressed. Tel: +1 212 241 7075; Fax: +1 212 289 4107; Email: Stuart.Sealfon{at}mssm.edu

Received June 1, 2005. Revised July 25, 2005. Accepted September 28, 2005.

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.


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