Anti aβ

Alpha-linoleic acid (cis-9,cis-12-octadecadienoic acid, Lot#30H8479) and Aβ_1–42 peptides were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. The antibodies used in the study included anti-Aβ (sc-28365), anti-p-Tau (sc-390476), anti-BACE-1 (sc-33711), p-Tau (sc-)anti-PSD-95 (sc-71933), anti-synaptosomal-associated protein 23 (SNAP-23) (sc-374215), anti-p-JNK (sc-6254), anti-Caspase 3 (sc-7272), anti-Bax (sc-7480), anti-Bcl-2 (sc-7382), anti-PARP-1 (sc-7008), anti-TNF-α (sc-52746), anti-p-NF-κB p65 (Ser536) (sc-136548), anti-TLR4 (sc-293072) anti-Iba-1 (sc-32725), anti-GFAP (sc-33673), and anti-β-actin (sc-47778) from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibodies were diluted in TBST (1:1000) (Santa Cruz Biotechnology) and the secondary anti-mouse HRP conjugated (Promega Ref# W402) and antirabbit HRP conjugated (Promega Ref# W401) antibodies that were diluted to 1:10,000 in 1 × TBST were obtained from Promega, USA. For confocal microscopic studies, the secondary fluorescent antibodies used were goat anti-mouse (Ref# A11029) and goat anti-rabbit (Ref# 2732), which were diluted in 1× phosphate-buffered saline (PBS).

The following antibodies were used in this study: anti-p-Akt, anti-p-NF-κB 65, anti-iNOS, anti-caspase-3, anti-PARP-1, anti-Bax, anti-Aβ, anti-BACE-1, anti-GFAP, anti-Iba-1, anti-pGSK3^ser9, and anti-β-actin (Santa Cruz Biotechnology), anti-Synaptophysin and anti-PSD95 (Cell Signaling).

Five microliters of Aβo, at a concentration of 50 μM, were applied to carbon-coated Formvar grids (Origen). Non-specific immunoreactivity was blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature and incubated with the primary antibody anti-Aβ (1:50; Santa Cruz Biotechnology) for 1 h. A secondary 5-nm gold-conjugated anti-mouse IgG antibody was used at a 1:20 dilution for 30 min. Samples were fixed with a 2% glutaraldehyde solution for 5 min. Aβo were stained with 5 μL of 0.2 % (wt/vol) phosphotungstic acid and the grid was air-dried. Samples were examined using a JEOL 1200 EX II electronic microscope.