Alexa fluor 488 conjugated goat anti chicken igy secondary antibody

Macromolecular-centered free radicals were detected upon stimulating splenocytes with 1 µmol/L BDC-2.5 mimotope in the presence of 1 mmol/L 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Dojindo) in tissue culture–treated chamber slides. Cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, blocked with 5% BSA in PBS, and incubated with 20 μg/mL of chicken IgY anti-DMPO as previously described (25 ,26 (link)). DMPO adducts were detected with Alexa Fluor 488–conjugated goat anti-chicken IgY secondary antibody (1:500; Invitrogen). P-STAT4 (Y693) and STAT4 were detected with Alexa Fluor 488–conjugated donkey anti-rabbit IgG (H+L) secondary antibody (1:500) and Cy3-conjugated Donkey anti-mouse IgG (H+L) (1:500) (Jackson Immunoresearch), respectively. CD4 T cells were identified with Alexa Fluor 647–conjugated streptavidin secondary antibody (1:500; Life Technologies). Images were obtained with an Olympus IX81 Inverted Microscope at a 40× objective and analyzed with cellSens Dimension imaging software, version 1.12. For quantitation of fluorescence intensity, three to six images were obtained for each data point. Each image was collected at the same exposure time, adjusted to the same intensity level for standardization, and the fluorescence intensity was measured using ImageJ software (National Institutes of Health).