Synapse density quantification

Hippocampal neurons were fixed at 13–15 DIV, in 4% formaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 5 min at room temperature. Cells were then washed three times in PBS, permeabilized and blocked for 30 mins in 5% FBS, 0.1% Triton X-100 in PBS. Primary antibodies diluted in 5% FBS in PBS were applied up to 2 h. Cultures were immunostained with vGLUT1 and Homer1, with Alexa-conjugated secondary antibodies and mounted using Prolong Diamond anti-fade reagent with DAPI (Invitrogen). 18–35 z-stack for each image (step size 0.35 μm) were acquired. Image analyses were performed with ImageJ superimposing stacks of each colour channel. The co-localization analysis was performed by evaluating the labelling of pre- and post-synaptic protein couples (vGLUT1/Homer1). Co-localization puncta with areas of 0.1–2 μm² were considered bona fide synaptic boutons and their number per field automatically counted evaluated by using the ImageJ colocalization plug-in.