Figure 2. Immunocytochemical labeling of adult human retinas

A: Retina treated with no primary antibody but with Cy-2- (green) and Cy-5- (blue) labeled secondary antibodies shows strong autofluorescence of lipofuscin granules in the retinal pigment epithelium (R). p, photoreceptor layer; n, inner nuclear layer; g, ganglion cell layer. B: Retina treated with polyclonal anti-PEDF (green) that had been pre-adsorbed with the immunizing peptide. Note absence of label in photoreceptors (p), inner nuclear layer (n) and ganglion cell layer (g). However, some photoreceptor outer segments (*) are weakly positive, suggesting that this label may not be due to authentic PEDF. C: Polyclonal anti-PEDF (green) labels photoreceptor outer segments and rod and cone cytoplasm. The interphotoreceptor matrix is labeled (blue) with anti-IRBP. Note PEDF-positive rod nuclei (r) but PEDF-negative cone nuclei (arrowheads). The ganglion cells (g) and some cells in the inner nuclear layer (n) are labeled (green) with pAb anti-PEDF, as are cells in the choroid (c). D: Polyclonal anti-PEDF (green) labels the inner segments of the cones, which are labeled throughout with mAb 7G6 (blue), producing a cyan color. One cone outer segment (cyan, arrowhead) is positive with both pAb anti-PEDF and mAb 7G6. E: Polyclonal anti-PEDF (green) labels all rod outer segments and one cone outer segment (arrowhead). Monoclonal anti-calbindin (red) labels the cytoplasm of red and green sensitive cones, but not the cytoplasm of blue sensitive cones, identifying the cone with the PEDF-positive outer segment as a blue cone. F: Monoclonal anti-PEDF (blue) labels the interphotoreceptor matrix, which is also positive with pAb anti-IRBP (green), producing a cyan color. p, photoreceptor layer; n, inner nuclear layer. Cell nuclei are stained (red) with propidium iodide. R, retinal pigment epithelium; p, photoreceptor layer; n, inner nuclear layer; g, ganglion cell layer. Bars = 40 mm.