Electron microscopy was performed as an independent approach to investigate the presence of amyloid fibrils in drusen.
Figure 6 shows three different drusen (
Figs. 6A–C, with higher magnification of the boxed area shown in
Figs. 6E–G, respectively) and associated sub-RPE deposits. The section shown in
Figures 6A and
6E was reacted with an antibody against vitronectin and visualized with a secondary antibody conjugated to 15-nm gold particles. Vitronectin reactivity appeared throughout the drusen and in adjacent sub-RPE deposits inside the RPE basal lamina. Of note, long strands of ∼10-nm diameter fibrils are abundant in these sub-RPE deposits (
Fig. 6E). Such fibril dimension and morphology is typical of amyloid fibrils.
17 Figures 6B and
6F show a section of a druse with sub-RPE fibrils reacted with the 6E10 anti-Aβ antibody followed by a secondary antibody conjugated to 25-nm gold particles. The 6E10 staining pattern appeared to be nonspecific inasmuch as the gold particles were seen distributed randomly throughout. Nevertheless, the diameter of the fibrils can be measured against the 25-nm gold particles. The fibril diameter also appeared to be ∼10 nm, consistent with that of amyloid fibrils. Some strands appeared to be twisted (
Fig. 6F, arrow), a morphologic characteristic often seen in amyloid fibrils.
Figures 6C and
6G are micrographs from a different druse with a similar pattern of amyloid fibrils in the sub-RPE deposits. To determine whether the sub-RPE fibrils may be formed by Aβ, we stained adjacent sections from a drusen specimen that contained sub-RPE deposits with WO (
Fig. 6D) and 6E10 (
Fig. 6H) antibodies. Both showed strong staining of vesicles that are more concentrated at the bottom right corner of the figures. Similar to that shown in
Figure 3, WO stained sub-RPE deposits in addition to vesicles (
Fig. 6D). Although 6E10 has the ability to bind to Aβ fibrils in senile plaques of AD brain (
Fig. 2), no reactivity was seen in the sub-RPE deposits (
Fig. 6H). These data suggest that amyloid fibrils in these sub-RPE deposits are not derived from Aβ and are likely to be formed by different proteins or peptides. Amyloid fibrils at the edge of vesicular structures were not identified by EM. A previous EM study of Aβ-stained drusen vesicles did not reveal obvious structures consistent with amyloid fibrils,
30 perhaps because the few vesicles examined by EM contained OC-reactive small fibril seeds rather than WO-reactive mature fibrils. Further studies of more of these vesicular structures are needed to address whether some vesicles are lined with EM-visible amyloid fibrils. Nevertheless, the current data obtained from immunofluorescence and EM clearly demonstrate the presence of bona fide amyloid fibrils in substructural elements of drusen and in sub-RPE deposits.