The anti-GluR1 antibody or the anti-GluR2/3 antibody was used at a concentration of 1–14 μg/ml, diluted in saline buffered with 0.1 M Tris and containing 0.1% Triton X-100 and 0.3% NaCl, adjusted to pH 7.4 (TBST-7.4). The ultrathin sections mounted on Formvar-coated 200-mesh grids were incubated in the primary antibody within 5–8 hours after ultrathin sectioning. The incubation in the primary antibody was overnight (~14 hours) at room temperature. Grids were immersed in droplets of TBST-7.4 containing either the anti-GluR1 or the anti-GluR2/3 antibody. On the following day, the grids were rinsed in TBST-7.4, and then rinsed in TBST adjusted to pH 8.4 and with 0.9% NaCl (TBST-8.4) before being incubated for 1 hour at room temperature in TBST-8.4 and containing the secondary antibody at a dilution of 1:40. At the end of this incubation in the secondary antibody, grids were rinsed first in TBST-7.4 in 0.9% NaCl and then in water, and postfixed for 10 minutes at room temperature, by using 1% glutaraldehyde in water. At the end of the PEG procedure, grids were briefly counterstained with Reynold’s lead citrate (15 seconds).
