Immunofluorescence Analysis of Lytic Reactivation

SLK.iBAC-GFP cells were induced with Dox (1 μg/ml) for 24 h to trigger lytic reactivation. The cells were then washed three times with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) (#DF0131, LEAGENE, Beijing, China) for 10 min. Next, the fixed cells were washed with PBS for three times, permeabilized with 1% Triton X-100 for 5 min, and blocked with 10% goat serum (Antgene, Wuhan, China) for 1 hour at room temperature. After washing with wash buffer (1X PBS with 0.1% Tween 20) for three times, the cells were incubated with rabbit anti-K-RTA or mouse anti-FLAG antibodies diluted in PBS containing 1% BSA at 4°C overnight. Cells were washed with wash buffer (PBS containing 0.05% Tween-20) and then incubated with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (1:1000; Invitrogen) and Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (1:1000; Invitrogen) at room temperature for 1 hour. After washing with wash buffer and ultrapure water, the slides were mounted with DAPI Fluoromount-G mounting medium (SouthernBiotech). Finally, the images were acquired with a laser scanning confocal microscopy (Leica Stellaris 5) and processed using Image J and Leica image browser [60 (link)].