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Journal of Biomedicine and Biotechnology
Volume 2012 (2012), Article ID 627602, 9 pages
http://dx.doi.org/10.1155/2012/627602
Methodology Report

High-Resolution Whole-Mount In Situ Hybridization Using Quantum Dot Nanocrystals

Department of Biological Sciences, University of Cyprus, 1678 Nicosia, Cyprus

Received 30 August 2011; Revised 29 September 2011; Accepted 3 October 2011

Academic Editor: P. Bryant Chase

Copyright © 2012 Andriani Ioannou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescent in situ hybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K. The use of QDs dramatically increased sensitivity of whole-mount in situ hybridization (WISH) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification. Therefore, this method offers significant advantages both in terms of sensitivity, as well as resolution. Specifically, the use of QDs alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos. It also offers new imaging possibilities, including intracellular localization of mRNAs, simultaneous multiple-transcript detection, and visualization of mRNA expression patterns in 3D.