Prolong diamond antifade dapi mounting solution

Latent and reactivated iSLK.KSHV 219 cells were washed 3 times with 1X PBS and then fixed with 4% paraformaldehyde (PFA) for 10min at room temperature (RT). For cell membrane permeabilization, cells were incubated with 0.05% Triton X-100 at RT for 5min. Then, cells were blocked with IFA blocking solution (1% BSA, 3.5% Goat Serum and 0.1% Tween 20 in 1X PBS) for 1 h at RT. The primary antibody was subsequently diluted in the blocking solution and incubated overnight at 4°C. Monoclonal antibodies were used for human HIF2α (Bethyl Laboratories), KSHV K8.1 (Santa Cruz Biotechnology), KSHV ORF57 (Santa Cruz Biotechnology) and Calnexin (Santa Cruz Biotechnology). The next day, cells were washed 3 times with 1X PBS and incubated with Alexa 647 or Alexa 555 conjugated secondary antibodies (Invitrogen) diluted in blocking solution for 1 h at RT. Lastly, the stained cells were washed 3 times with 1X PBS, rinsed once with ddH₂O and slides were mounted using prolong diamond antifade DAPI mounting solution (ThermoFisher Scientific). Samples were analyzed using a Leica DMI6000B microscope with LASX software (Leica).