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 |
Externí odkaz: |