Figure 2.

Localization of pScop2 in the double retina of Pecten. (A) In situ hybridization in cryosections of isolated, fixed retinae incubated with digoxigenin-labeled sense or antisense riboprobes. After hybridization, transcripts were detected using AP-conjugated anti-DIG antibody. Left: The antisense-treated retinae show selective staining in the distal layer (DR), where the ciliary photoreceptors are located, but no labeling in the proximal retina (PR), where microvillar photoreceptors are found. Right: No staining at all appeared in the retinae incubated with the sense probe. (B) Western blot of retinal lysate using anti-pScop2 antibody. Proteins separated by SDS-PAGE (8%) before transferring to nitrocellulose membrane. A prominent band is visible near 50 kD, the predicted weight of pScop2. (C) Immunohistochemistry to localize pScop2 in eye slices; secondary antibodies were conjugated to Alexa Fluor 546. The lefthand panels show Nomarski images at two magnifications, and the righthand panels show the corresponding fluorescence microscopy images. Immunofluorescence is confined to discrete cells of the distal retina. DIC, differential interference contrast. (D) Immunocytochemistry in isolated cells. The retina was enzymatically dissociated, and the cell suspension was plated onto concanavalin A–treated coverslips before incubation with antibodies. The two classes of photoreceptors are recognizable by their distinct morphology; only the ciliary photoreceptors are immunopositive for anti-pScop2 antibody and display an accumulation of the opsin in the region of the ciliary appendages.