Immunocytochemistry/Immunofluorescence analysis of nNOS (neuronal) (phospho S1417) (green) showing staining in the cytoplasm and nucleus of SH-SY5Y cells treated with 100 ng/ml nocodazole (left) and untreated SH-SY5Y cells (right). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab5583 in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.