Murine T Cells were fixed in cold methanol for 1 min. After permeabilization with fixation/permeabilization buffer (BD Bioscience) for 1 h, the cells were blocked with 5% bovine serum albumin for 1 h. The cells were incubated with rabbit anti-FoxO1 and mouse anti-IKKβ primary antibodies (1:200 dilution) for 16 h and then incubated with anti-rabbit IgG-CF488 and anti-mouse IgG-CF594 secondary antibodies (1:500 dilution), respectively, for 1 h. The secondary antibodies were purchased from Biotium. The fluorescence signals were analyzed using Leica TCS SP5II confocal microscope 13 (link), 28 (link).
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