Figure 6. Close association of extracellular RGR-d and C5b-9 complement complex in Bruch’s membrane. A: The RPE-choroid section from a 78-year-old male donor was incubated first with DE21 antibody and anti-rabbit IgG conjugated to FITC and subsequently with C5b-9 monoclonal antibody and anti-mouse IgG antibody conjugated to Texas Red. (Top panels) RGR-d aggregates, rounded bodies or speckles in Bruch’s membrane, basal deposit (D), and intercapillary space are observed. (Middle panels) Specific labeling of C5b-9 in the intercapillary regions and along the walls of the capillaries coincides to a large extent with that of RGR-d. (Bottom panels) Significant co-localization of RGR-d and C5b-9 is shown with DAPI counterstain in the merged images of top and middle panels, as indicated by the arrows. B: Merged image of representative negative control with DAPI counterstain performed in parallel by omitting both primary antibodies. There is a complete absence of immunostaining in Bruch’s membrane, choroid, intercapillary regions, and basal deposits. Autofluorescence of lipofuscin granules is visible in RPE cells. Scale bar, 10 μm.