Ioannou A et al. (2012), High-resolution whole-mount in situ hybridizati...

XB-ART-45467

J Biomed Biotechnol 2012 Jan 01;2012:627602. doi: 10.1155/2012/627602.

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

Ioannou A, Eleftheriou I, Lubatti A, Charalambous A, Skourides PA.

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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.

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Species referenced: Xenopus
Genes referenced: a2m actc1 actl6a ltbp1 myod1 pkm tbxt xa-1

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	Figure 1. Proteinase K treatment is necessary to allow QD penetration and labeling of deep tissues in Xenopus and QD705â€‰nm anti-Dig conjugates can be used for the detection of transcripts in wholemount in situ hybridization experiments. (a) Detection of biotinylated 4G10 (anti-Phosphotyrosine) antibody using streptavidin conjugated 655â€‰nm QDs in a triton permeabilized embryo. Specific staining can be seen at the cell-cell boundaries of the ectodermal cells of the fin (upper part of the image) but not in the deep tissues (somites at the bottom part of the image). (b) Detection of biotinylated 4G10 (anti-Phosphotyrosine) antibody using streptavidin conjugated 655â€‰nm QDs in a PK permeabilized embryo. Specific staining of the deep intersomitic boundaries can be seen. Superficial cells of the fin cannot be seen due to degradation of this delicate structure by the PK treatment. (c) QD705â€‰nm anti-DIG antibody labeling of the probe for LTBP1 generates a staining pattern that closely matches the published expression for this mRNA [1]. QDs label the somites as well as anterior neural and neural crest tissues including the branchial arches and a region surrounding the eye. (d) Imaging of the QD labeling for LTBP1 in the somites at 10X magnification.
	Figure 2. QD-streptavidin staining of LTBP1 compares favorably with the published staining achieved using standard chromogenic protocols in the deeper structures of the embryo [1]. (a) A transverse section from a whole mount in situ indicating the LTBP1 transcript expression pattern using QD705â€‰nm streptavidin. The somitic staining obtained using QDs is identical to the published data using the chromogenic protocol [1], showing that the QD-streptavidin solution can penetrate and stain the deep areas of the somites. (b) A series of transverse optical sections of a QD-streptavidin-stained embryo for the LTBP1 message. The optical sections reveal that the staining previously identified as notochord by the chromogenic protocol is in fact somitic mesoderm flanking the notochord (nc: notochord, sm: somatic mesoderm).
	Figure 3. In situ hybridization using QDs compares favorably with chromogenic in situ hybridization staining for a number of well-characterized mRNAs. (a) 705â€‰nmQD-streptavidin staining for amylase on dissected Xenopus guts, using a biotinylated amylase probe, is compared to the staining obtained by chromogenic reaction (left). The staining using QDs is identical to that using a chromogenic reaction and restricted to the pancreas, where amylase mRNA is expressed. It is worth noting that the pancreas, a morphologically identifiable organ, is extremely autofluorescent making detection of fluorescent staining difficult. (b) Comparison of the QD and chromogenic staining for MyoD a muscle marker (using a FITC-labeled probe). The 655â€‰nmQD anti-FITC and the chromogenic staining are similar, but the QD staining gives much better resolution of the posterior somites. (c) Comparison of QD versus chromogenic staining for the Edd transcript, an endodermal marker expressed through the tadpoles gut at varying levels (using a Biotin-labeled probe). The staining using a chromogenic protocol is significantly stronger in this case and the 705â€‰nmQD-Streptavidin seems restricted to the high expression regions. Careful observation reveals that the staining is present throughout the gut region but is masked by the intense autofluorescence of the gut. (d) Comparison of the QD staining versus the chromogenic staining for Xbra, a widely used mesodermal marker (using a Biotin-labeled probe). The marker is known to label the mesodermal belt at gastrula stages; both the chromogenic, as well as the QD-streptavidin protocols result in the same staining pattern consistent with the mesodermal belt.
	Figure 4. Double whole-mount in situ hybridization against cardiac actin and Xa-1 and the intracellular distribution of LTBP1 and Xa1. (a) A comparison of the staining pattern between the chromogenic and QD-based visualization of the FITC-labeled Xa-1 probe, shown in red, reveals that the QD staining is identical to that obtained using the standard chromogenic protocol. (b) An embryo processed using Biotin and FITC-labeled probes against cardiac actin (green) and Xa-1 (red), respectively. The two probes were visualized with spectrally resolvable QDs demonstrating that two color fluorescent in situs can be performed using QDs. The inset shows the chromogenic staining for cardiac actin for comparison. (c, d) Images of embryos processed for whole-mount in situ hybridization and counterstained with Hoechst (blue) at 20X (c) and 40X (d) magnification, showing differences in the intracellular distribution of the transcripts of Xa-1 (c) and LTBP1 (d), both shown in red.

References [+] :

Akerman, Nanocrystal targeting in vivo. 2002, Pubmed