Ab3267

Deparaffinized sections were stained with Hematoxylin and Eosin (Medical Chemical Corporation, 310-787-6800). For IHC, tissue sections were deparaffinized at 60 °C for 30–45 min and rehydrated followed by heat-induced antigen retrieval for three minutes. Sections were blocked using 5% bovine serum albumin and 0.1% Triton X-100 diluted in PBS for 30 min at room temperature and immunostained overnight with primary antibodies against TRA-1-85 (1:100; MAB3195, R&D Systems), RPE65 (1:500; AB105366, Abcam) and Rhodopsin (1:400; AB3267, Abcam) at 4 °C. Sections were stained with secondary antibodies goat anti-mouse IgG conjugated with rhodamine (1:100; 115 025 146, Jackson Immunoresearch) and goat anti-rabbit IgG conjugated with FITC (1:00; 115 095 144, Jackson Immunoresearch) for one hour at room temperature. Sections were mounted with fluorescein-enhancing mounting medium with DAPI (Vector Laboratory) and imaged using an Ultraviewer ERS dual spinning disk confocal microscope (PerkinElmer) equipped with a C-Apochromat 10 × high dry lens, a C-Apochromat 40 × water immersion lens NA 1.2 and an electron multiplier cooled digital camera (CCD). Images were captured and processed using PerkinElmer Volocity imaging software.

Primary antibodies used were mouse monoclonal antibody against HA tag (ab18181), rabbit polyclonal antibody against Myc tag (ab9106), mouse monoclonal antibody against rhodopsin (ab3267) (Abcam, Cambridge, MA, USA), and rabbit polyclonal antibody against PRCD [5 (link)]. Secondary antibodies used were peroxidase-conjugated AffiniPure goat anti-mouse IgG, peroxidase-conjugated AffiniPure goat anti-rabbit IgG, Cy3-conjugated donkey anti-mouse IgG, Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and GST HRP rabbit polyclonal IgG (Santa Cruz Biotechnology, Dallas, TX, USA).