Fitc conjugated goat anti mouse antibody

Cells were washed three times with PBS and treated with 1% Triton X-100. Next, cells were incubated with the anti-p65 antibody (Abcam, 1 : 200) at 4°C overnight and then with an FITC-conjugated goat anti-mouse antibody (Cell Signaling Technology, 1 : 100) at 37°C for 1 h. Cells were counterstained with DAPI (Beyotime Biotechnology, Shanghai, China) to visualize the nuclei.

To examine the morphological changes, parent and recurrent cells on cover slips were fixed with methanol: acetic acid (2:1) at −20 °C for 10 minutes then washed with 1X PBS and permeabilized with 0.5% Triton X-100 for 15 minutes on ice. After subsequent washing, the cells were incubated for one hour at 37 °C in 5% BSA solution (Sigma Aldrich), followed by overnight incubation with β-actin (mouse; 1:500; Sigma Aldrich) and ATRX (rabbit; 1:750; Abcam) at 4 °C. After washing thrice for 5 min with PBS, coverslips were incubated with FITC conjugated goat anti- mouse antibody (1:100; Cell Signalling) for 45 min. Nuclei were counterstained with DAPI (0.5 μg/mL) for 1 minute, washed thrice with 1X PBS and mounted using VECTASHIELD mounting media (Vector Labs). The cells were visualized under Zeiss LSM 510 Meta Confocal Microscope.