Figure 4.

Background-free immunofluorescence staining via CDR. (a-d) Representative fluorescence intensity maps obtained after a 1-hour incubation with (a) 7 nM QDot-1'Ab (1× [probe]) under rotary shaking and (b) 0.3 nM (0.04× [probe]), (c) 0.9 nM (0.13× [probe]), and (d) 1.5 nM (0.21× [probe]) QDot-1'Ab using CDR procedure. Images were normalized and color-coded with a heat map. Scale bar, 250 μm. (e) Average fluorescence intensities of Lamin A staining achieved after a 1-hour incubation under rotary shaking with 1× [probe] and CDR with 0.04×, 0.13×, and 0.21× QDot-1'Ab concentration. Consistent with qualitative observations in (a-d), quantitative analysis demonstrated that comparable staining could be obtained with ~8-times lower probe concentration via CDR methodology. Error bars represent one standard deviation of an average Lamin A staining intensity from four different fields of view. (f) A strong background fluorescence originating from the QDots in 1× [probe] bulk solution used for staining under rotary shaking required extensive specimen washing prior to imaging. At the same time, 0.13× [probe] solution employed with CDR methodology featured nearly no background, enabling real-time monitoring of staining evolution. Scale bar, 50 μm.