EM immunocytochemistry was performed to discriminate the synaptic versus nonsynaptic localization of GluR1 subunits forming the GluR1-containing AMPARs (summarized in Fig. 3), by using the postembedding immunogold labeling (PEG) procedure (Aoki et al., 2000). The same procedure was used to detect the synaptic versus nonsynaptic localization of GluR2 and -3 subunits forming AMPARs. The PEG procedure was as described by Phend et al. (1995). Ultrathin sections were collected on grids from all animals within a single 3-hour period, and then processed in parallel, so as to reduce interanimal and intertissue variability resulting from the immunocytochemical procedure. Seventy to 90-nm-thick ultrathin sections were collected on 200-mesh Formvar-coated nickel grids. We collected one to three ultrathin sections per grid and at least three grids per sample for GluR1 immunocytochemistry and at least three additional grids for GluR2/3 immunocytochemistry. Of these, at least one underwent a no-primary-antibody control of the PEG procedure (described in the next section), and the remaining grids served as duplicates to immunolabel for the GluR1 or the GluR2/3 subunits of AMPARs.
