Alexa fluor 594 red conjugated goat anti mouse antibody

Different U-2OS cell sublines were grown in 24-well plate for 48 h and fixed in 3.7% buffered paraformaldehyde. After permeating with methanol for 20 min, the cells were blocked with 1% BSA in PBS for 1 h. Subsequently, the cells were incubated with Pgp antibody (dilution: 1 : 50) at 4 °C overnight. After washing three times with PBS, the cells were then incubated with Alexa Fluor 594 (Red)-conjugated goat anti-mouse antibody (Invitrogen, Carlsbad, CA, USA) in a dilution of 1 : 1000 for half an hour. The nuclei were stained with 1 μg ml⁻¹ Hoechst 33342 (Life Technologies). Images were obtained by Nikon Eclipse Ti-U fluorescence microscope (Nikon Instruments, Inc., Melville, NY, USA) equipped with a SPOT RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).

The ovarian cancer cell lines were seeded into 24-well plates at a concentration of 2 × 10⁴ cells/ml for 72 h and fixed in 4% paraformaldehyde for 15 min at room temperature. Following fixation, the cells were washed in 1× PBS (3 times, 5 min each) prior to permeabilization with 100% ice-cold methanol in a −20°C refrigerator for 10 min. After blocking with 5% goat serum for 1 h, the cells were then incubated with the primary antibodies ATR (1:200, Cell Signaling Technology), p-ATR (1:200, Abcam), and β-Actin (1:1000, Sigma-Aldrich) overnight at 4°C in a humidified chamber. The next day, we removed the primary antibody solution and rinsed before incubation with fluorochrome-conjugated secondary antibody for 1 h at room temperature in the dark. The secondary antibodies Alexa Fluor 488 (Green) conjugated goat anti-rabbit antibody and Alexa Fluor 594 (Red) conjugated goat anti-mouse antibody were purchased from Invitrogen (NY, USA) and diluted in 5% goat serum at 1:1000. Finally, they were washed and incubated with a 4′,6-diamidino-2-phenylindole (DAPI) solution (1:10,000) for 5 min. Pictures were obtained with a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., Melville, NY, USA) equipped with a SPOT RTTM digital camera.