Immunofluorescence of CFH, Fib3 and controls in normal and dry AMD eye GCL (ganglion cell layer), INL (inner nuclear layer), ONL (outer nuclear layer), RPE (retinal pigment epithelium), CH (choroid).

<p>Nuclei (gray) and autofluorescence from lipofuscin granules (magenta) are visible in all cryosections. Scale bar = 50µM. <b>A:</b> Section of retina from a normal human eye labeled with mouse monoclonal antibody against Fib3, mFib3 (green) and an anti-CFH antibody (red). In normal retina, the Fib3 immunoreactivity is present in the ganglion cell layer as reported previously. CFH is restricted to choroidal blood vessels. <b>B</b>: Section from H/H AMD eye (#57985) with soft druse. CFH (red) is detected in globules within the druse. <b>C:</b> Similar section to B labeled for both mFib3 (green) and an anti-CFH antibody (red). Strong Fib3 immunoreactivity is observed in the soft druse (arrows) and the signal colocalizes with that of anti-CFH. Merged colors vary according to relative staining for both antibodies. <b>D:</b> The colocalization of Fib3 and CFH in soft drusen (arrows) is confirmed with a newly generated anti-fib3 antibody-rFib3 (rabbit polyclonal antibody raised against the N-terminal region of human Fib3 (blue). The two independently derived anti-Fib3 antibodies show identical staining patterns. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068088#pone.0068088.s003" target="_blank">Fig. S3</a>). <b>E:</b> Little labeling of either Fib3 or CFH was observed in a hard druse (*) from the same AMD donor (left panel). <b>F:</b> Control antibodies for markers for retinal glia (anti-GFAP rabbit polyclonal, blue) and rod photoreceptors (anti-rhodopsin mouse monoclonal, green) do not label CFH-positive areas within the soft drusen (arrows). <b>G–I:</b> Detailed region of a similar soft druse from H/H AMD donor #68536. <b>G:</b> green label for Fib3; <b>H:</b> red label for CFH; <b>I:</b> merged image. Arrows point out some of the deposits showing similar levels of labeling for both antibodies. Scale bar = 50µM.