Quadruple fluorescence: Cy2 + Cy3 + Cy5 immunolabelling and Hoechst DNA staining, in a sensory ganglion. S-100 protein in glial cells surrounding neuronal perikarya and many axons (Cy3, YELLOW). Many perikarya and large-diameter axons react for neurofilament proteins (Cy5, RED), the peptide CGRP is found in a subpopulation (Cy2, GREEN). Cy3 was shown through a yellow-channel filter set, for maximum signal yield and high selectivity. Cy5 was observed directly, and photographed, through a visual filter set. Note complete separation of the four labels: Cy2-avidin, Cy3-avidin, Cy5-avidin, and bisbenzimide-DNA (BLUE: narrow-band filter set).
See: Ferri et al., Cell Vision, 1998. Image: Gian-Luca Ferri, M.D.
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