Rabbit anti sox2

Cells were seeded on 18 mm round coverslips in the 12-well plates, grown for 72 h in the absence or presence of p32-I, incubated with fixation buffer (Biolegend) for 20 min at RT, permeabilized and blocked for 45 min. Fixed cells were incubated in primary antibodies for 3 h at RT, washed and incubated with secondary antibodies for 1 h. Samples were washed and incubated with DAPI (Invitrogen) for 20 min at RT in the dark. Coverslips containing stained samples were mounted onto glass slides using FluorSave ™ reagent (Calbiochem). Primary antibodies used: rabbit anti-TOM20 (Santa Cruz, 1:1000 dilution), mouse anti-MYC (Santa Cruz, 1:250 dilution). Rabbit anti-Nestin (Proteintech, 1:50 dilution), rabbit anti-Sox2 (Millipore, 1:100 dilution). Secondary antibodies used: Alexa Fluor 488 and Alexa Fluor 594 (Invitrogen). Samples were analyzed using Leica TCS SP8 Inverted Microscope.

For immunostaining cells were fixed for 15mins at 4°C in 4% paraformaldehyde in phosphate buffer saline (PBS), then washed in PBST (PBS with 0.1% Triton-X100). The cells were blocked for 30mins at RT using blocking buffer A (1% BSA/0.1% Triton-X100 in PBS). Cells were incubated with primary antibodies o/n at 4°C and with secondary antibodies at room temperature for 2 hours. For sequential immunostaining with two goat antibodies after the end of the 1st immunostaining, cells were incubated with blocking buffer B (1% goat serum, 1% BSA, 0.1% Triton-X100 in PBS) for 1h at RT. The second goat antibody was directly conjugated with a fluorophore (Bra–NL557). Cells were incubated with the goat-conjugated antibody for 2h at RT in blocking buffer B. After washing with blocking buffer A the cells were mounted with DAPI containing Prolong Antifade (Molecular Probes). The following primary antibodies were used: goat anti-Brachyury (1:500) (R&D), goat anti- BraNL557 (1:250) (R&D), goat anti-Tbx6 (1:200) (R&D), rabbit anti-Sox2 (1:500) (Millipore), mouse anti-Tuj1 (1:1000) (Covance), mouse anti-Hoxc10 (1:200) (Abcam). The fluorescent images were captured using an inverted Leica SP5 confocal microscope.

Primary antibodies used were: mouse anti-GFAP (Thermo Fisher Scientific), mouse anti-nestin (Rat-401), mouse anti-p62 (Abcam), rat anti-phosphorylated p62 (MBL: D343-3), mouse anti-ubiquitin (Santa Cruz Biotechnology), rabbit anti-TBK1 (Cell Signaling), rabbit anti- phosphorylated TBK1 (Novus Biologicals), rabbit anti-GFAP (Dako), rabbit anti-Ki67 (Spring Bioscience), rabbit anti-p62 (Enzo), rabbit anti-Sox2 (Millipore), rat anti-Ki67 (BioLegend), and guinea pig anti-doublecortin (anti-DCX; EMD Millipore). Secondary antibodies were goat anti–rabbit IgG-FITC, goat anti–rabbit IgG–Texas red, goat anti–mouse IgG-FITC, goat anti–mouse IgG–Texas red, goat anti–mouse IgG-HRP, and goat anti–rabbit IgG-HRP (Jackson Immunology). Dihydroethidium (DHE) was purchased from Sigma-Aldrich and Amlexanox was purchased from MedChemExpress. Transfections were carried out using Lipofectamine 3000 reagent (Invitrogen).

hNPCs were fixed in formaldehyde 3.7% for 15 min at RT and permeabilized in 0.2% Triton X-100 for 15 min Primary antibodies were incubated overnight in 2% BSA at 4 °C, following 40 min of 2% BSA blockage. After washing with PBS, secondary antibodies were incubated for 1 h at RT in the dark. Cells were washed three times with PBS and nuclei were stained with DAPI. Coverslips were mounted on slides using Aqua-Poly Mount (Polysciences) whereas cells on the 384 well plates were covered with glycerol and sealed with AlumaSeal CS (Excel Scientific) for image acquisition in confocal microscopy (Leica) and Operetta (Perkin Elmer), respectively. Primary antibodies used: mouse anti-MAP2 (Sigma-Aldrich), mouse anti-Ki-67 (BD Biosciences), rabbit anti-PAX6 (Santa Cruz Biotechnology), rabbit anti-GFAP (Dako), rabbit anti-γ-H2AX (Cell Signaling Technology), rabbit anti-FOXG1 (Abcam), rabbit anti-DYRK1A (Sigma-Aldrich), rabbit anti-SOX2 (Millipore), mouse anti-β-tubulin III (Millipore), mouse anti-nestin (Millipore), rabbit anti-TBR2 (Millipore). Secondary antibodies used: goat anti-mouse Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific). Data are expressed as relative protein expression in comparison with basal protein expression in control with vehicle (DMSO).