A subset of tissue obtained from naïve, paired, and unpaired conditions were processed for preadsorption control. For these, the GluR1 primary antibody was preadsorbed with a control peptide corresponding to the antigen used to generate the antibody. The original Western blot report on the nature of the AMPAR antibodies indicated that preadsorption of antibodies was successful at a control peptide concentration of 1 mg/ml (Wenthold et al., 1992). Therefore, the control peptide concentration was set to a value as close as possible to this original condition, namely, 800 μg/ml; lower concentrations of the control peptide were also tried. For all conditions, pre-adsorption was achieved by preincubating the antibody with the control peptide, together with 0.05% sodium azide, overnight at room temperature and for an additional hour at 30°C, before the antibody-peptide mixture was applied to grids.
