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

Figure 3

In situ hybridization using QDs compares favorably with chromogenic in situ hybridization staining for a number of well-characterized mRNAs. (a) 705 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 655 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 705 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.