Anti rhodopsin antibody

Retina and RPE/choroid flat mounts, and embedded sections, were deparaffinized and processed for antigen-epitope retrieval. Samples were incubated in a streamer autoclave at 120°C for 10 min in antigen retrieval solution (Histofine, Nichirei Biosciences) and then allowed to cool. Sections were then incubated with protein-blocking serum-free solution (Dako). For double staining, the anti-RPE65 and anti-CRALBP antibodies (Calbiochem) were added to the slides and incubated overnight at 4°C. We also stained additional sections with the following antibodies: anti-4 hydroxynonenal antibody (catalog #STA-035, Cell Biolabs), anti-rhodopsin antibody (catalog #ab3267, Abcam), and anti-ZO-1 antibody (catalog #40-2300, Thermo Fisher Scientific). After washing the slides the following day, the secondary antibodies Alexa Fluor-labeled (594 nm) and red goat anti-rabbit IgG (Molecular Probes) were added and incubated at room temperature for 1 hr. Slides were washed, and anti-fade reagent with DAPI (Prolong Gold antifade reagent with DAPI, Molecular Probes) was added. Pictures were taken within 24 hr using a fluorescence microscope (Leica). Confocal imaging was taken by using an Olympus Fluroview unit and an Olympus BX50 confocal microscope (Olympus).