Mouse anti tau

Cortices were microdissected from E18.5 embryos, digested in trypsin (Pierce), manually triturated and plated on 35-mm well onto poly-D-lysine coated coverslips at a density of 150,000 cells for the neurite outgrowth assay or 500,000 for cellular localization experiments. Cells were plated in medium containing DMEM with 10% FBS and 1% penicillin/streptomycin and 4 h after plating, media was completely removed and replaced with Neurobasal media containing B-27+ (Gibco/Invitrogen), 1% penicillin/streptomycin, and L-glutamine. Cells were cultured for 24 h or one week in a 37°C incubator containing 5% CO₂, then fixed for 10 min in 4% paraformaldehyde. Immunocytochemistry was conducted using the following antibodies: mouse anti-Tau (1:200; Abcam), rabbit anti-MAP2 (1:1000; Abcam), rabbit anti-Mllt11 (1:300, Abcam), rabbit anti-Tuj1 (Tubb3; 1:1000, Biolegend), and mouse anti-acetylated α-tubulin (1:1000, Sigma).

The primary antibodies used for immunocytochemistry in this study were rabbit anti-Aβ42 (1:35, Millipore), mouse anti-NES (nestin) (1:500, R&D), rabbit anti-OCT4 (1:500, Abcam), mouse anti-SYP (synaptophysin) (1:500, Abcam), mouse anti-TRA-1-81 (1:250, BD Pharmingen), and rabbit anti-TUBB3 (tubulin β3 class III) (1:1000, Covance). The secondary antibodies included Alexa594-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa488-conjugated donkey anti-goat IgG (1:200, Invitrogen), and Alexa488-conjugated donkey anti-mouse IgG (1:200, Invitrogen). The antibodies used for western blotting were mouse anti-GSK3β (glycogen synthase kinase 3β) (1:500, Cell Signaling Technology), mouse anti-phospho-GSK3β (Ser9) (1:500, Cell Signaling Technology), mouse anti-tau (1:500, Abcam), mouse anti-phospho-tau (Ser396) (1:1000, sigma), mouse anti-phospho-tau (Thr181) (1:500, Cell Signaling Technology), and mouse anti-TUBB3 (1:10000, Biolegend). The secondary antibodies used included goat anti-mouse IgG-HRP (horseradish peroxidase) (1:20000, GE Healthcare), goat anti-rabbit IgG-HRP (1:20000, GE Healthcare), and donkey anti-goat IgG-HRP (1:2500, Santa Cruz). The immune complexes were detected using an enhanced chemiluminescent substrate (ECL, Biotools).