Alexa fluor 555 conjugated anti rabbit igg fab2

U937 cells were seeded in Lab-Tek II Chamber Slides (Thermo Fisher Scientific, USA), and differentiated with PMA as previously described. Cells were treated with inhibitors for 1 h, and then infected with T. cruzi for 30 min. After infection, cells were washed and fixed in 4% paraformaldehyde (PFA) for 15 min at RT and blocked with 5% bovine serum albumin in PBS containing 0.1% Triton X-100 for 2 h. Cells were then incubated with monoclonal anti-p65 antibodies (Cell Signaling, USA #8242) overnight at 4°C. The samples were washed with PBS and incubated with Alexa Fluor Ⓡ 555-conjugated anti-rabbit IgG Fab2 (Cell Signaling, USA #4413) for 1 h. Finally, nuclei were stained with DAPI for 5 min, and cells were mounted with Dako Fluorescence Mounting (Dako, USA). The cells were imaged using a Nikon Eclipse 400 epifluorescence microscope (Nikon, Japan), and images were analyzed by mean intensity using ImageJ software (ImageJ 1.47v).