Synaptic Density Quantification of Cultured Neurons

Synaptic density of cultured neurons was measured by counting PSD95 and vGlut1-labelled clusters attaching to neuronal dendrites and presented as the number of synapses per micron of dendrite as previously described67 (link). Briefly, after fixation in 4% paraformaldehyde for 30 min, neurons were blocked in 5% goat serum for 30 min at room temperature. PSD95, vGlut1 and MAP2 were labelled by rabbit anti-PSD95 (Cell Signaling, #3450, 1:200), guinea pig anti-vGlut1 (Synaptic System, #135304, 1:400) and mouse anti-MAP2 (Sigma-Aldrich, M4403, 1:400) followed by goat anti-rabbit IgG conjugated with Alexa 594 (Invitrogen, A11037, 1:500), goat anti-guinea pig IgG conjugated with Alexa 647 (Invitrogen, A21450, 1:500) and goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, A11029, 1:500), respectively. Images were collected under a Nikon confocal microscope followed by three-dimensional reconstruction by using Nikon-Elements advanced Research software68 (link). The synapses were defined by colocalization of vGlut1 and PSD95. Dendritic segments between 20 and 50 μm from the soma were used for the analysis69 (link).