Immunostaining was performed as described earlier (33 (link), 34 ). Briefly, cells were cultured on four well glass chamber slides (Fisher Scientific, Rochester, NY) and pre-treated with 4μ8C (IRE1α inhibitor) 1h before S. aureus challenge. Following stimulation, cells were washed three time with PBS and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with an ethanol: acetic acid mixture (2:1) at −20°C for 10 min. and washed. The fixed cells were blocked in 1% (w/v) BSA for 1h at room temperature followed by incubation with primary antibodies (1:100 dilution) overnight at 4°C. Cells were washed with PBS and incubated with specific fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200 dilutions) for 1h at room temperature. Following incubation cells were washed with PBS and mounted in Vectashield anti-fade mounting medium with DAPI (Vector Laboratories). Slides were visualized using an Eclipse 90i fluorescence microscope (Nikon, Melville, NY)