Rabbit anti tubb3

Immunolabeling was performed as previously described [26] (link). Cells were fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature followed by washes with PBS. After incubating in blocking solution containing 10% FBS, 2% goat or donkey serum, 0.1% Triton X-100, 0.02% sodium azide, fixed cells were incubated overnight at 4°C with the following primary antibodies: rabbit anti-GFP (1∶200, Invitrogen, used to detect YFP-expressing cells throughout the study), rabbit anti-Pax6 (1∶200, Millipore), rabbit anti-neurofilament 145 (NF145)(1∶750, Millipore), rabbit anti-Tubb3 (1∶100, Covance), rabbit anti-Otx2 (1∶50, Abcam), goat anti-Sox2 (1∶50, Santa Cruz Biotechnology), mouse anti-Pou4f1 (1∶100, Millipore), mouse anti-Crx (1∶100, Abnova), mouse anti-NF68 (1∶400, Sigma), mouse anti-Islet1 (1∶5, Developmental Study Hybridoma Bank). After washing extensively with 0.1% Tween in PBS, the cells were incubated with secondary antibodies conjugated with Alexa 488 or Alexa 594 (1∶500, Invitrogen). Fluorescent images were captured using a Nikon E800 microscope equipped with a SPOT II camera or an Olympus FluoView 1000 confocal microscope.

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).