293T (Positive) and HL-60 (Negative control) cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with ZO-1 Antibody( bs-1329R) at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).