Dab reaction

Except where indicated, staining was done on 40-μm free-floating sections of a one-in-six series through the entire hippocampus and cortex. Tissue was blocked in 10 % normal serum from the species in which the secondary antibody was raised, with the addition of 0.1 % Triton X-100. Primary antibodies were diluted in 3 % normal serum with 0.1 % Triton X-100, and tissue incubated overnight at 4 °C. Biotinylated secondary antibodies were diluted in 3 % normal serum with 0.1 % Triton X-100 and were incubated for 2 h at room temperature. Enzyme detection was done using avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA, USA) followed by color development in diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA). Antibodies and dilutions were as follows: rat CD11b (Serotec, Raleigh, NC, USA; 1:500); mouse CD68 (Serotec; 1:200); rabbit YM1 (Stem cell technology, Vancouver, BC, Canada; 1:400); rat Marco (Serotec; 1:500); mouse NeuN (Millipore, Temecula, CA, USA; 1:1000). After incubation for 24–48 h with appropriate primary and biotinylated secondary antibodies, tissue was treated with Vectastain ABC reagent (Vector Labs) and visualized with DAB reaction (Sigma-Aldrich). Controls included omitting primary or secondary  antibodies.

2–4 month-old wild-type mice (n = 6 per group) or APP/PS1 mice (n = 3 per group) were treated with 8 h or 14 h, respectively, of AMPA or artificial CSF via reverse microdialysis then immediately transcardially perfused with ice-cold phosphate buffer saline (PBS) with 0.3% heparin. Brains were removed, fixed in 4% paraformaldehyde for 24 h at 4 °C, then placed in 30% sucrose prior to freezing and sectioning. Coronal brain sections 50 μm wide were sliced in 300 μm intervals using a freezing sliding microtome. Sections were then immunostained to visualize astrocytes or microglia using antibodies against glial fibrillary acidic protein (GFAP; 1:500, ThermoFisher) as an astrocytic marker or against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Wako Laboratory Chemicals, Richmond, VA) as a microglial marker. Biotinylated secondary antibody, horseradish peroxidase-conjugated streptavidin, and DAB reaction (Sigma) were used to develop. Brain sections were imaged with a Nanozoomer slide scanner (Hamamatsu Photonics, Bridgewater, NJ). Staining density was qualitatively evaluated by blinded observers and vehicle- and AMPA-treated groups were compared. Images shown are representative.

Histological sections of 5 µm were also performed for immunostaining procedures of samples included in paraffin blocks. Endogenous peroxidase was blocked with 3% hydrogen peroxide/10 mM PBS pH 6.0 for 15 min. Afterwards, the sections were microwaved in 3% milk buffer for 30 min and incubated overnight with primary antibodies; anti-Bax (DAKO A/S Denmark; N-20 5c493, rabbit polyclonal), anti-Bcl-2 (DAKO A/S Denmark; N-19 5c492, mouse polyclonal), anti-Ki67 (DAKO A/S Denmark; F0788, mouse monoclonal) with dilutions of 1∶600, 1∶150 and 1∶500 respectively at 20°C. The sections were incubated with post primary block and secondary antibodies (Novocastra Laboratories Ltd; Novolink RE 7260-K) for 1 h, and processed using the DAB reaction (0.5 mg/ml, Sigma, USA, St Louis). Any cell type showing cytoplasmic staining was considered positive for quantitative analysis [11] (link), [24] (link), which was performed by a blinded observer (C.B.D.). The microscope and the software used to capture images for quantitative analysis were the same as those used for the hematoxylin and eosin study.