Figure 2. Bassoon localization at the rod photoreceptor ribbon complex is more stable than RIBEYE localization. A, B: Representative images of the outer plexiform layer (OPL) with the photoreceptor ribbon synapses double labeled for RIBEYE (magenta) and Bassoon (green) from untreated (A) and EGTA-treated (B) retinae. Horseshoe-shaped RIBEYE lines the horseshoe-shaped Bassoon in untreated retina (A). In EGTA-treated retina, RIBEYE often disintegrates into several aggregates (B). C, D: Immunoelectron micrographs of rod photoreceptor ribbon synapses from EGTA-treated retina. RIBEYE labeling (C) is found at the spherical material pinching off the ribbon (arrowhead) as well as at the active zone (arrows). In contrast, Bassoon labeling (D) is concentrated at the ribbon base (arrows) and the material pinching off the ribbon is not stained for Bassoon (arrowhead). E: Quantification of the Bassoon/RIBEYE co-localization in untreated and EGTA-treated retinae. In double stained vertical sections, either horseshoe-shaped (HS) RIBEYE or horseshoe-shaped Bassoon profiles were counted and checked for the morphological appearance of the respective other marker. When counts were based on horseshoe-shaped Bassoon (bar groups 1 to 3), the fraction of sites where RIBEYE staining appears in a horseshoe shape decreases under EGTA conditions, whereas the punctate (club-shaped, CS, or spherical-shaped, SS) and cloudy appearance of RIBEYE staining increases concomitantly. When counts were based on horseshoe-shaped RIBEYE (bar group 4), only a negligible fraction of sites had no horseshoe-shaped Bassoon under both conditions. The total number of horseshoe-shaped Bassoon sites in EGTA-treated retinae was reduced in the same OPL volume. F, G: Representative images of the OPL double labeled for the rod photoreceptor terminal marker Veli3 (magenta) and Bassoon (green) from untreated (F) and EGTA-treated (G) retinae. In the EGTA-treated retina, Veli3 labeled rod photoreceptor terminals often show punctate or a lack of Bassoon labeling, which is in contrast to the horseshoe-shaped Bassoon labeling present in rod photoreceptor terminals of untreated retina. Scale bars: 5 µm (A, B, F, G), 0.2 µm (C, D).