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The coverslips were then washed with PBS and the cells were fixed with 4% PFA for 15 min, washed with 0.05% Tween 20 (Promega, WI, USA) diluted in PBS (PBST), permeabilized with 0.2% Triton X-100 (Promega, WI, USA) for 10 min, and blocked with 3% BSA in PBST for 3 h