Rabbit anti ha tag 3724

AAV8.CMV.HsPEX1.HA or AAV8.CMV.EGFP was added to 84-31 cells (a modified HEK293 cell line) at a MOI of 10⁵ or 5 × 10⁵ (10 μL or 50 μL of 10¹⁰ vg/μL per 2 × 10⁶ cells at time of viral expression). Cells were harvested after 48 h, lysed, and separated on a 4%–12% Bis-Tris gradient gel (Invitrogen, Waltham, MA, USA) and transferred to nitrocellulose membrane. Membranes were blocked in 5% milk and hybridized in 2% milk with 1:1,000 rabbit anti-HA tag (3724; Cell Signaling Technology, Danvers, MA, USA), 1:1,000 rat anti-GFP (04404-84; Cedarlane, Burlington, ON, Canada), and 1:15,000 rabbit anti-human β-tubulin (ab6046; Abcam, Cambridge, MA, USA), followed by appropriate horseradish peroxidase (HRP)-conjugated secondary antibody and visualized by enhanced chemiluminescence (ECL) using an Amersham 600 Imager. Mouse retina immunoblotting followed the same method, with the addition of 1:1,000 rabbit anti-HsPEX1 (13669-1-AP; Proteintech, Rosemont, IL, USA).

For cryosections, eye cups from PBS-perfused mice were fixed 3 h in 10% formaldehyde; incubated in 10% (30 min on ice), 20% (1 h on ice), and 30% (4°C overnight) sucrose in 0.1 M PB; and then embedded and frozen in frozen-section compound (VWR, Mississauga, ON, Canada). 5 μm retinal cryo-sections were blocked (1% normal goat serum, 0.1% Triton X-100, 10% bovine serum albumin [BSA] in PBS) for 1 h, washed, incubated at 4°C overnight with primary antibody in incubation buffer (0.1% Triton X-100, 10% BSA in PBS), washed, incubated 90 min with secondary antibody, and washed. Coverslips were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, Burlington, CA, USA), and retinas were visualized using a Leica DMI600 microscope with a DFC345FX camera and LASX software (Leica, Richmond Hill, ON, Canada). The primary antibody used was 1:300 rabbit anti-HA tag (3724; Cell Signaling Technology, Danvers, MA, USA).

Eyes were fixed in 4% formalin for 5 min at room temperature, a hole made in the cornea with a needle (27G), and fixed for additional 25 min. Eyes were sectioned at the limbus and anterior segments discarded. The posterior eye cups were collected and the neural retina detached from RPE/choroid/sclera. Neural retina flatmounts were incubated in 0.1% Triton X-100, 10% fetal bovine serum (FBS) in PBS (saturation buffer) for 45 min, and then overnight at 4°C with primary antibody or fluorescein peanut agglutinin (Vector Laboratories, Burlingame, CA, USA) in saturation buffer. RPE/choroid/sclera flatmounts were treated with PBS solution containing 0.1% Triton X-100 for 45 min and then incubated overnight at 4°C with primary antibody and/or TRITC phalloidin (ECM Biosciences, Versailles, KY, USA) for RPE cell-counter staining. IF was analyzed and images acquired using a Zeiss LSM780 laser-scanning confocal microscope. The primary antibodies used were rabbit anti-HA tag (3724; Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-human cone arrestin (AB15282; Millipore, Burlington, MA, USA). Alexa Fluor 488- and 594-conjugated antibodies were used as secondaries (Invitrogen, Waltham, MA, USA).