Figure 8.

Relative intensity and separation of fluorophores. Low-sensitivity methods for immunofluorescence detection using a Cy2-tagged secondary antibody usually only gives a signal in the green channel. However, when such signals are amplified, for example using a tyramine-based procedure, not only are strong signals evident in the green emission channel (panel A), there is often sufficient “bleed-through” fluorescence in the tail of the emission range of the green fluorophore now to give a signal that is detected also as red (panel B, here using very specific Texas Red filters), and the image appears as if it is double-labeled, even though in this case only a single, Cy2-tagged antibody has been used. A further control for this is to reverse the order of the red and green fluorophores, using the weaker antigen as green, and the stronger one as red. Note the highly suspicious morphologic identity of the 2 images.