Af488 conjugated goat anti mouse

Following experimental treatment, BV2 cells cultured in chambered microslides were fixed by incubation in 2% formaldehyde in PBS for 10 min at 4 o C, washed and non-specific antibody binding was minimised by incubation for 30 min at room temperature in PBS containing 10% FCS and 0.05% Triton X-100 (all Thermofisher Scientific, UK). Cells were then incubated with rabbit anti-mouse p67Phox monoclonal antibody (1:500, clone EPR5064, Abcam Ltd, Cambridge, UK) and mouse anti-mouse gp91phox monoclonal antibody (1:50, clone 53, BD Biosciences, UK) overnight at 4°C in PBS with 1% FCS and 0.05% Triton X-100. Cells were washed and incubated with AF488-conjugated goat anti-mouse and AF647-conjugated goat anti-rabbit secondary antibodies (both 1:500, Thermofisher Scientific, UK) in PBS with 1% FCS and 0.05% Triton X-100 at room temperature for 1 hr. Cells were washed with PBS, nuclei were defined by incubation with 180 nM DAPI in ddH2O for 5 min, and cells were mounted under Mowiol mounting solution. Cells were imaged using an LSM710 confocal microscope (Leica, UK) fitted with 405nm, 488nm and 647nm lasers and a 63x oil immersion objective lens (NA 1.4mm, working distance 0.17mm). Images were captured with ZEN Black software (Zeiss, Cambridge, UK) and analysed with ImageJ 1.51w (National Institutes of Health, USA).