Gar af488

For experiments shown in Figures 3C, 3D, 4A, 4B, and 5D, live neurons were washed in HBSS based imaging solution (see Fluorescence lifetime imaging section above) for 5min, fixed in 4% paraformaldehyde (PFA) in HBSS for 10min. PFA reaction was then quenched by 5min incubation in 0.1M Glycine in PBS. Neurons were placed in a blocking/permeabilization solution containing 2% goat serum and 0.1% Triton X-100 in PBS for 30min, followed by immunostaining with primary antibodies (1/100 in the blocking/permeabilization solution for PSD-95 or P-Thr320-PP1 and PP1γ; overnight at 4°C). Samples were washed 3 times in PBS before secondary antibodies were applied (1/1000 of GAM-AF647 and/or GAR-AF488 for 1h at room temperature, from Life Technologies). After 3 finals washes in PBS, samples were mounted in Prolong Gold mounting media (Life Technologies). Neurons expressing the indicated constructs (identified with nuclear-mCherry expression) were imaged on an Olympus FV-1000 confocal microscope with a 60X oil immersion objective. Software recommended filters were used for each dye to acquire z stacks with a 0.5 μm separation.