Figure 5.

The zs-4 antibody recognizes the extracellular domain of Crb2a. (A-A") Single confocal z-section of the photoreceptor layer in a 6 d wild-type retina labeled with rabbit panCrb antibodies (A) and zs-4 monoclonal (A') and the two channels merged (A"). The arrow indicates where anti-panCrb and zs-4 labeling does not appear to fully overlap. (B-C") Confocal z-projection of a 6 d retina labeled with anti-HA antibody (monoclonal IgG₃; red) and zs-4 antibody (blue) and merged with anti-GFP (green). (B-B") Wild-type retina. (C-C") Crb2a^Extra_TM transgenic retina. Endogenous Crb2a labeling is weak in comparison to Crb2a^Extra_TM protein (C"). (D-F') Confocal z-projection of the photoreceptor layer in a 6 d retina labeled with zs-4 antibody (red), rods express GFP (green). TO-PRO-3 (blue) labeling reveals the position of nuclei in the photoreceptor layer. (D-D') Wild-type retina. (E-E") Crb2a^Extra_Secr transgenic retina. Single z-section from the white boxed area in E', showing zs-4 labeling around the inner segment (indicated by yellow bracket and outlined in yellow) of a double-cone, the cell-body is circled in white (E"). (F-F') Crb2a^Extra_Secr transgenic retina where no rods are present. (G-H) Single z-section of the brain ventricular region of a 40 hour postfertilization embryo labeled with zs-4 antibody (red) showing ventricular surface labeling. Cell nuclei are labeled with TO-PRO-3 (blue). (G) Wild-type embryo. (H) ome (crb2a) mutant embryo. The midline (where the ventricles would normally form) is indicated by the white arrow, and eyes indicated by dashed white lines. Scale bars, 5 μm (A-A"', D-F') 10 μm (G, H).