Immunofluorescence analysis was performed as previously described (Posada-Duque et al. 2013 (link)). Briefly, the cells were incubated overnight at 4°C with the following primary antibodies: mouse PSD95 (1:250, Calbiochem), rabbit PSD95 (1:250, Cell Signaling) and mouse MAP2 (1:2000, Sigma). The Alexa-594 fluorescent antibody was used as a secondary antibody (1:1000, Molecular Probes). The nuclei were stained with Hoechst 33258 (1:5000, Invitrogen), and the cells were incubated with phalloidin conjugated with Alexa 594 (1:2000, Molecular probes) for 1 h. The cells were washed 4 times in buffer, coverslipped using Gel Mount (Biomeda), and observed under an Olympus IX 81 epifluorescence microscope or a DSU Spinning Disc Confocal microscope. No staining was observed when the primary antibodies were omitted.
XY images were collected using an Olympus IX 81 microscope with 10× (NA, 0.4), 40× (NA, 1.3) or 60× (NA, 1.42) oil-immersion objectives. Z-stack images were collected at 0.4-µm intervals on an Olympus IX 81 DSU Spinning Disc Confocal microscope (60× oil-immersion; NA, 1.42). The image stacks were deconvolved using CellM imaging software (Olympus).
XY images were collected using an Olympus IX 81 microscope with 10× (NA, 0.4), 40× (NA, 1.3) or 60× (NA, 1.42) oil-immersion objectives. Z-stack images were collected at 0.4-µm intervals on an Olympus IX 81 DSU Spinning Disc Confocal microscope (60× oil-immersion; NA, 1.42). The image stacks were deconvolved using CellM imaging software (Olympus).