Alexa fluor 488 goat anti mouse secondary antibody

10,000 cells per chamber were seeded onto eight-chamber slides with 0.5 ml of appropriate cell culture media. The next day, appropriate samples were treated with camptothecin (0.5–2 µM final) for 2 h. Cells were then washed and fixed with 4% PFA for 10 min, followed by two washes. Chamber walls were them removed, and three final washes were conducted using 0.01% Triton X-100 in PBS. The whole slide was then blocked with 5% BSA in PBS–Triton X-100 for 1 h at RT. Following three more washes, the slide was incubated with S9.6 antibody (1:1,000 in 5% BSA–PBS–Triton X-100) overnight at 4°C. The next day, following washes, the slide was incubated with Alexa Fluor 488 goat antimouse secondary antibody (ab150113; Abcam) and DAPI (both 1:1,000 in 5% BSA–PBS–Triton X-100) for 1 h in the dark. The slide was kept in the dark through three more washes and dried by aspiration, and a coverslip was applied with a few drops of VECTASHIELD mounting medium (H-1000; Vector Laboratories) and sealed with clear nail polish. Slides were then imaged using the Nikon Eclipse 80i fluorescence microscope (Nikon Instruments) equipped with the CoolSNAP HQ2 camera (Photometrics) at 60× magnification with a plan apochromatic 60×/1.40 NA oil objective. Images were subsequently analyzed using ImageJ software.