Ab80592

Protein samples were prepared in Laemmli sample buffer (2% SDS, 10% glycerol, 60 mM Tris–HCl pH6.8) and heated for 10 min at 95 °C. Sample proteins were quantified using Bicinchoninic acid assay (BCA) reagent (Pierce) according to manufacturer instructions. Prior to loading, samples were supplemented with 100 mM dithiothreitol and 0.005% bromophenol blue. Following SDS-PAGE electrophoresis and western blotting, TDP2 and KU80 were detected using a rabbit anti-TDP2 primary antibody (Thomson et al. 2013 (link)), and KU80 was detected as a loading control using an anti-Ku80 rabbit monoclonal primary antibody (Abcam Ab80592). For overexpressed mCherry-tagged TDP2, anti-mCherry antibody (Abcam ab167453) was employed.

In vivo assessment was performed by spectral domain optical coherence tomography (SD-OCT) (Envisu, Bioptigen) 1 week post transplantation surgery. Histological and immunohistochemical analysis was also performed. Enucleated eyes were cryopreserved according to previously published protocols (Cuenca et al., 2018 (link)). The tenon’s capsule and extraocular muscles were removed as much as possible, and a cut was made around the limbus with a scalpel blade to allow the fixative solution to enter into the eye cavity. Eyes were fixed in 4% paraformaldehyde for 2 h at room temperature. After washing with 0.1 M sodium phosphate buffer (pH 7.4), eyes were immersed in increasing concentrations of sucrose (up to 25%) before freezing. Cryosections (12 mm thickness) were collected and processed for immunofluorescent labeling with anti-Ku80 antibody (rabbit anti-human, 1:200, ab80592, Abcam), and melanosome antibody (mouse anti-human, 1:200, HMB45, Dako). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear counterstaining (Molecular Probes). Fluorescence images were acquired with a confocal microscope (C2, Nikon).

Cells were fixed with 4% formaldehyde (BioSharp, BL539A) for 30 min at room temperature. Then, cells were permeabilized with 0.5% Triton X‐100 for 5 min. Fixed cells were blocked in 5% BSA for 1 h at room temperature and probed with H3K9me3 (CST, 1:1000, #13969), H3K9ac (CST, 1:400, #9649), RAR gamma (Abcam, 1:1000, ab191368), and KU80 (Abcam, 1:1000, ab80592) antibodies in blocking solution overnight at 4 °C. Then, cells were washed in PBS and incubated with Alexa‐Fluor‐594‐conjugated secondary antibody (Abcam, 1:1000, ab150080) and DAPI (Biosharp, BS097) for 2 h under dark conditions. Images were acquired with a Leica SP8 (Wetzlar, Germany, 63× objective at 1024 × 1024 resolution with 2 times zoom) confocal microscope.