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

Autor: Ioannou, Androulla, Eleftheriou, Iro, Lubatti, Andrea, Charalambous, Anna, Skourides, Paris A.
Přispěvatelé: Skourides, Paris A. [0000-0003-3502-5729]
Jazyk: angličtina
Rok vydání: 2012
Předmět:
Health
Toxicology and Mutagenesis
Xenopus
Nanoparticle
High resolution
lcsh:Medicine
whole mount in situ hybridization
nanocrystal
sensitivity analysis
biotin
antibody
animal
genetics
cellular distribution
In Situ Hybridization
Fluorescence
messenger RNA
digoxigenin
nanoparticle
article
imaging
quantum dot
methodology
General Medicine
staining
Fluorescence
Enzymatic amplification
Molecular Medicine
Endopeptidase K
Biotechnology
oligonucleotide
lcsh:Biotechnology
proteinase K
embryo
prenatal development
In situ hybridization
Biology
chemistry
Permeability
animal tissue
fluorescence analysis
lcsh:TP248.13-248.65
Quantum Dots
Genetics
gene expression profiling
streptavidin
Animals
RNA
Messenger
Molecular Biology
fluorescence in situ hybridization
Fluorescent Dyes
nonhuman
Oligonucleotide
Methodology Report
Gene Expression Profiling
lcsh:R
Molecular biology
Nanocrystal
Quantum dot
Biophysics
Nanoparticles
fluorescent dye
in situ hybridization
permeability
metabolism
Zdroj: Journal of Biomedicine and Biotechnology, Vol 2012 (2012)
Journal of Biomedicine and Biotechnology
J.Biomed.Biotechnol.
ISSN: 1110-7251
1110-7243
Popis: The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescentin situhybridization 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-mountin situhybridization (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.
Databáze: OpenAIRE
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