Figure 3.

Direct observation and analysis of subretinal flecks. White subretinal flecks were observed in a Mfrp^174delG mouse by fundus photography (see Fig. 2C). (A) Eyes were then dissected to remove the cornea, iris, and lens. White spots were still visible through the retina with a dissecting microscope (black arrowheads). (B) After the retina was removed, the white structures remained adherent to the RPE and were more clearly imaged (white arrowheads). These structures were absent in regions where the RPE was peeled off during dissection, exposing the underlying choroid (asterisk). (C) The tissue was then processed and embedded in plastic for electron microscopy. One-micrometer sections revealed that the structures were clearly cellular in nature (arrowhead). (D) When viewed at the electron microscope level, we observed that the cells contained pigment granules and small, circular, membranous structures. (E) Frozen sections of eyes with retinas removed were probed with an RPE65 antibody (green), which labeled RPE but not the adjacent subretinal cells (arrowheads). Note that nuclei were stained with Hoechst (blue). (F) Flat-mounted eyecups with retinas removed were used to test for immunoreactivity with macrophage marker F4/80 (green). Subretinal cells in this preparation were highly autofluorescent (red), making them easy to identify. (G) F4/80⁺ cells in wild-type eyecups were sparse and much less autofluorescent. mv, microvilli; ch, choroid; POS, photoreceptor outer segment.