Round glass coverslip

For indirect immunofluorescence, cells were fixed on round glass coverslips (Fisher Scientific) with chilled methanol. After incubation with the primary antibodies and the respective secondary antibodies for 50 min each, coverslips were washed with PBS and mounted with antifade mounting solution (Invitrogen). A fluorescence microscope (Leica DM 2500) was used to obtain representative images.

Primary cell cultures of DRG neurons were conducted as previously described [23 (link)]. Thoracic and lumbar DRGs were dissected from control and Ano1/Adv^fl/fl mice, and collected in cold culture medium (4°C) containing a mixture of DMEM and F-12 solution, 10% fetal bovine serum (Gibco BRL), 1 mM sodium pyruvate, 50–100 ng/ml nerve growth factor (Alomon, Jerusalem, Israel), and 100 units/ml of penicillin/streptomycin. Ganglia was washed with culture medium and incubated for 30 min in a warm (37°C) DMEM/F-12 mixture containing 1 mg/ml of collagenase (Type II; Worthington Biomedical). DRGs were then washed 3 times with Mg²⁺- and Ca²⁺-free Hank’s solution, and incubated while gently shaking in Hank’s solution containing 2.5 mg/ml of trypsin (Roche Diagnostics) for 30 min at 37°C. The trypsin-containing solution was then centrifuged at 100 g for 10 min. The pellets obtained after centrifugation were washed gently 2–3 times with culture medium and gently triturated with a fire-polished Pasteur pipette. Cells were plated onto round glass coverslips (Fisher), which had been previously treated with poly-L-lysine (0.5 mg/ml), in small Petri dishes (35 × 12 mm). Cells were then placed in a 37°C incubator in a 95% air/5% CO₂ atmosphere. Cells were used 2–4 days after plating.

Cells were seeded on round glass coverslips (Fisherbrand) and treated according to each individual experimental setup. They were then fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at room temperature. All the reagents mentioned below were prepared based on the immunofluorescence (IF) protocol provided by Cell Signaling Technologies. Fixed samples were blocked for 1 h at room temperature in IF blocking buffer (1X PBS, 5% normal serum and 0.3% Triton X-100). Afterwards, cells were reacted overnight at 4 °C with the appropriate primary antibody diluted in the IF antibody dilution buffer (1X PBS, 1%, BSA and 0.3% Triton™ X-100). Cells were washed three times in 1X PBS and then incubated with the appropriate secondary antibody in IF antibody dilution buffer for 1 h at room temperature. Cells were finally washed three times using 1X PBS and were mounted on glass slides in Dako containing 0.1 µg/mL Hoechst 33342 (Sigma-Aldrich). Imaging was performed using a LSM800 confocal microscope (Zeiss) or a Leica TCS SP8-DLS (TCS SP8 laser scanning confocal microscope with DLS light sheet module).

The immunofluorescence was performed on the fixed cells grown on the round glass coverslips (Thermo Fisher Scientific, USA) in 35 mm cell culture dishes. The cells were incubated with primary antibody against H2AX (Abcam, ab195188,1:50) overnight at 4 °C, followed by rhodamine-conjugated anti-mouse secondary antibodies incubation for 1 h, and DAPI (Beyotime Biotechnology, China) as a nuclear stain. The cells were then examined under confocal fluorescence imaging microscope (TCSSP5; Leica, Mannheim, Germany).