cells with human anti-nuclear antigen (ANA) antibodies, followed
by biotinylated anti-human IgG and QD-streptavidin. The nuclear
antigens were clearly labeled with red QD 630–streptavidin (Fig. 4A).
In control cells incubated with secondary antibodies and QD-
streptavidin alone, no detectable signal was observed (Fig. 4B).
We have noticed that whereas staining of cell surface antigens in
whole cells and tissue sections is reliable and effective, staining of
cytoplasmic and nuclear structures is more variable. Some QD probes
showed strong positive signals with low background; others exhibited
weaker positive staining or higher background. This variation might
be caused by inconsistencies in the surface coating or bioconjugation
of QDs, and we are investigating the underlying mechanism.
To further assess the specificity of the QD-streptavidin conjugates,
we labeled nuclear antigens with QD 630–streptavidin and microtubules with QD 535–streptavidin in the same experiment by means
of sequential biotin-streptavidin binding. When examined under an
epifluorescence microscope with a 460 nm short-pass excitation filter (ex 460SP) and a 500 nm long-pass emission filter (em 500LP),