High-resolution whole-mount in situ hybridization using Quantum Dot nanocrystals.

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.