Goat anti p22phox

Dissociated neurons were fixed with a 4% paraformaldehyde/4% sucrose mixture in PBS that was warmed to 37°C in advance for 10 min at room temperature. The neurons were then permeabilized by incubation with a 0.1% (vol/vol) Triton X-100 solution in PBS for 10 min at room temperature and washed gently with PBS. The neurons were blocked with 1% BSA in PBS for 1 h at room temperature and incubated at 4°C overnight with primary antibodies, including rabbit anti-PV (1:600; Abcam), goat anti-gp91^phox (1:200; Santa Cruz Biotechnology), goat anti-p22^phox (1:200; Santa Cruz Biotechnology), and mouse anti-PSD95 (1:200; Chemicon), diluted in 1% BSA. After the neurons were washed three times with PBS, they were incubated with secondary antibodies, including goat anti-rabbit IgG-FITC (1:300; Santa Cruz Biotechnology), goat anti-mouse IgG-Cy3 (1:600; Bioworld Technology) and donkey anti-goat IgG-Cy3 (1:800; Abcam), for 1 h at room temperature. After the neurons were washed three times with PBS, they were incubated with DAPI to label nuclei. Fluorescence images were obtained by confocal scanning microscopy (Leica, TCS SP2, Germany). For a given sample, 5–10 images, which were taken at 1-μm intervals, were collapsed to generate a projected image. The number of PSD95-positive puncta was measured by Image J (National Institutes of Health, Bethesda, MD, USA).