Sc 32323

The following primary antibodies and reagents were used: mouse anti-Olig2 (MABN50, Millipore), mouse anti-SUMO1 (33-2400, Thermo Fisher Scientific), mouse anti-BrdU (sc-32323, Santa Cruz), mouse anti-p53 (sc-126, Santa Cruz), rabbit anti-p53 (ab32389, Abcam), mouse anti-Flag (F1804, Sigma), mouse anti-Myc (sc-40, Santa Cruz), rabbit anti-Olig2 (AB9610, Millipore), rabbit anti-SUMO1 (4940, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (9661, Cell Signaling Technology), rabbit anti-gamma H2AX (phospho S139) (ab2893, Abcam), rabbit anti-Ki67 (ab16667, Abcam), mouse anti-phosphoserine (ab6639, Abcam), rabbit anti-Olig2 (phospho S10 + S13 + S14) (ab183487, Abcam), rabbit anti-Histone H3 (4499, Cell Signaling Technology), rabbit anti-acetyl-p53 (Lys379) (2570, Cell Signaling Technology), rabbit anti-p21 (ab218388, Abcam), rabbit anti-HA (H6908, Sigma), and SUMOylation 1 Affinity Beads (ASM11, Cytoskeleton). Etoposide (ETO, E1383), temozolomide (TMZ, T2577), BrdU (B5002), and tamoxifen (TAM, T5648) were purchased from Sigma. CHIR-99021 (S2924) was purchased from Selleck.

At E14.5 and E15.5, pregnant females received intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU at 5 mg/mL and 20 μl/g of mouse), 2 hours before euthanasia. Embryo heads were harvested, fixed overnight in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4°C, soaked in 30% sucrose in PBS until the tissues sank to the bottom of the tube, and embedded in Optimal Cutting Temperature (OCT) compound. Frozen 12 μm sections were collected on Fisherbrand superfrost plus slides using a cryostat. Slides were stained with Hematoxylin and Eosin (H&E), with antibodies to Pax6 (PRB-278P; 1/500 dilution; Covance, Emeryville, CA, USA) or BrdU (sc-32323; 1/1000 dilution; Santa Cruz, Santa Cruz, USA), as previously described [33 (link)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed using the ApoTag Kit (EMD Millipore) following manufacturer’s instructions.

MEFs were isolated from E9.5 embryos of each genotype and cultured in DMEM with 10% FBS. Cells were pulsed for 1 hour with 10μM bromodeoxyuridine (Brdu) and fixed in 4% PFA for 20 minutes at room temperature. After fixation, cells were double stained with anti-BrdU (Santa Cruz sc-32323, 1:400) and anti-cleaved caspase-3 primary antibodies (Cell Signaling #9661, 1:250). Nuclei were stained with DAPI (SIGMA, 0.5 μg/ml). Photographs were taken using a Nikon TE300 microscope.

To characterize the proliferation of supporting cells, BrdU staining and immunofluorescence assays were conducted as follows. The larvae at 72 hpf were placed in 10 mM BrdU solution (Sigma-Aldrich, #B5002-5G, Saint Louis, MO, USA) for 24 h at 28.5 °C. After washing in embryo medium to remove the BrdU, the larvae at 96 hpf were fixed overnight in 4% PFA at 4°C and permeated with 1% Triton X-100 for 0.5 h at RT. Then, the larvae were immersed in 2 N HCl for 0.5 h at 37 °C followed by blocking with 10% donkey serum for 1 h at 37 °C. After that, the primary antibodies of a chicken polyclonal anti-GFP (1:500 dilution, Abcam, #ab13970), a rabbit polyclonal anti-SOX2 (1:500 dilution, Abcam, #ab97959) and a mouse monoclonal anti-BrdU (1:500 dilution, Santa Cruz Biotechnology, #sc-32323, Dallas, TX, USA) were used to label the HCs, supporting cells and proliferative cells, respectively. At last, the secondary antibodies of a Alexa Fluor^TM 488 goat anti-chicken lgG (H + L) (1:1000 dilution, Invitrogen, #A-11039), a Alexa Fluor^TM 555 donkey anti-rabbit lgG (H + L) (1:1000 dilution, Invitrogen, #A-31572) and a Alexa Fluor^TM 647 donkey anti-mouse lgG (H + L) (1:1000 dilution, Invitrogen, #A-31571) were used to detect the primary antibodies, respectively.

Cells were prepared on poly-l-lysine-coated glass cover slips. 10 μM BrdU (Yeasen, 40204ES60) in Dulbecco’s phosphate-buffered saline was added 4 h before fixation. After fixing in 4% PFA at 4 °C overnight, cells were denatured in 2 N HCl at room temperature for 30 min and then rinsed in 0.1 M borate buffer (pH 8.0, 300 μl/well) for 10 min. After incubation in blocking solution (PBS containing 10% normal goat serum and 0.3% TX-100) for 1 h, cells were then treated with mouse anti-BrdU antibody (1:200; SC-32323, Santa Cruz) overnight at 4 °C. After a wash stage with PBS, cells were incubated with donkey anti-mouse (conjugated to Alexa-Fluor 576) secondary antibodies for 2 h at room temperature. Washed three times in PBS, cells were mounted using mounting solution (Prolong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI); P36931, Life Technologies). The slides were imaged using a fluorescence microscope (Olympus IX70, Olympus America, Inc., Center Valley, PA, USA). Five fields per well were randomly selected for cell counting.