Mouse anti oct4

Immunostaining and WB were performed as described (Chen et al., 2019 (link)). The following antibodies were used: mouse anti-Oct4 (Millipore), goat anti-Sox2 (Santa Cruz), mouse anti-Tra1-81 (Millipore), mouse anti-Tra1-60 (Millipore), mouse anti-Nestin (Chemicon), rabbit anti-Tuj1 (Covance), mouse anti-Tuj1 (Sigma), mouse anti-MAP2 (Sigma), rabbit H3K27me3 (Santa Cruz), rabbit anti-Pax6 (Covance), mouse anti-GABA (Sigma), mouse anti-Nkx2.1 (Millipore), rabbit anti-Tbr1 (Chemicon), mouse anti-vGluT1 (Millipore). MeCP2 antibody used in Western blot was a gift from Dr. Keping Hu.

Cells on coverslips were washed twice with PBS (5 min each), fixed with 4% PFA at room temperature (RT) for 10 min, then rewashed twice with PBS (5 min each). Coverslips were incubated for 1 h at RT in PBS, 0.03% Triton X-100, 3% BSA and 2% normal donkey serum (NDS, Jackson laboratory, #017-000-121) for permeablization and blocking non-specific antibody binding. Following that, incubation of primary antibodies was often carried out overnight at 4°C in the same buffer. Cells were then washed (3 times, 10 min each) in the same buffer and incubated with secondary antibodies (Jackson Laboratory) for 1 h at RT. Cells were finally washed with PBS (3 times, 10 min each) and mounted on glass slide. Staining was viewed and analyzed with Olympus up-right fluorescent microscope (BX51) or with confocal microscope (Leica TCS SP5 II). The following antibodies were used: mouse anti-Oct4 (Millipore), goat anti-Sox2 (Santa Cruz), mouse anti-Nestin (Chemicon), mouse anti-MAP2 (Sigma), rabbit anti-Synapsin (Synaptic Systems), mouse anti-vGlut1 (Synaptic Systems), Rabbit anti-GABA (Sigma).