Alexa488 goat anti rabbit igg

Cells were seeded in 6-well plates or 12 mm circular coverslips in 12-well plates. After treatment with the corresponding compound, fresh media was aspirated and cell monolayers were washed with PBS for 5 min three times. Cells were fixed with 4% paraformaldehyde for 15 min at 4 °C and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were rinsed with PBS three times, blocked with blocking solution (1% BSA, 10% goat serum in 0.1% PBS-Tween) for 1 h and stained with the primary antibody in blocking solution for 16 h at 4 °C. After PBS washing, cell monolayers were stained with the secondary antibody diluted in blocking buffer for 1 h at room temperature in darkness. Cell nuclei were stained with DAPI (Sigma-Aldrich) at a final concentration of 0.1 µg/mL for 15 min at RT. The primary antibodies targeted H2AX histone phosphorylation at Ser 139 (γ-H2AX; 1:200; Merck Millipore, Massachusetts, USA), ATM phosphorylation at Ser 1981 (1:200; Santa Cruz Biotechnology, Dallas, USA), DNA-PK phosphorylation at Ser 2056 (1:200; Abcam, Cambridge, UK), and Chk-1 phosphorylation at Ser 345 (1:50; Cell Signaling, Danvers, USA). The secondary antibodies were Alexa488 goat anti-mouse IgG (1:1000; Abcam) and Alexa488 goat anti-rabbit IgG (1:1000; Abcam). Fluorescence was quantified by high-content screening (INCell 2200, General Electrics, New York, USA).

BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y₁₂ (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times. Then, cells were stained with Alexa-488 goat anti-rabbit IgG (1:500; Abcam, UK) at 37°C for 1h. After washing with HBSS for three times, images were obtained by 535 nm emission filter based on an Olympus IX51 inverted fluorescence microscope with 40× oil objective.