Ab216324

MEFs transduced with lentiviruses and selected with antibiotics as described above were seeded at the concentration of 4 × 104 cells/well in the 4-well cell culture chamber slides (Nunc Lab-Tek Chamber Slide, C6932), and the cells were cultured for 48 hours under the presence of 3TC (10 μg/ml) or DMSO. Then the cells were washed with ice-cold PBS, fixed on ice with 4% paraformaldehyde (PFA) for 10 minutes, and then incubated in 100% methanol at −20 °C overnight. The cells were then treated with 200 mg/ml RNase A (Thermo Fisher) at 37°C for 4 hrs. Cells were blocked with 3% BSA and incubated overnight at 4°C with primary antibodies diluted in 3% BSA. The antibodies used and dilutions were as follows: Rabbit anti-mouse L1 Orf1p (Abcam, ab216324, 1:100), Mouse anti-single strand DNA (Sigma-Millipore, MAB3299, 1:100). After incubation, cells were washed three times with PBS for 5 minutes, and incubated with secondary antibodies (Goat anti-Rabbit IgG Alexa Fluor 488, Thermo Fisher, A11008, 1:1000; Goat anti-Mouse IgG Alexa Fluor 647, A21235, 1:1000) for 1 hour at room temperature, washed three times with PBS, counterstained with DAPI (Sigma-Millipore, MBD0015), washed, and coverslips were mounted on slides with ProLong Gold (Invitrogen). The cells were observed, and images were acquired with Leica SP8 on HC PL APO 40x/1.30 oil-immersion.