Mouse anti brdu

Frozen sections were incubated in 0.6% H₂O₂ for 15 min at room temperature and washed three times with PBS. For immunohistochemical staining, mouse anti-BrdU (1:150, Roche, Basel, Switzerland), rabbit anti-Ki67 (1:1000, Novocastra, UK), or guinea pig anti-DCX (1:2000, Millipore, Billerica, MA, USA), diluted in PBS containing 0.3% normal chicken serum and 0.3%Triton X-100, were used as the primary antibodies and incubated overnight at 4 °C. The sections were washed three times for 10 min each with PBS, incubated in biotinylated anti-mouse, anti-rabbit or anti-guinea pig IgG (1:250, Vector Laboratories, Burlingame, CA, USA), and then avidin-biotinylated enzyme complex (ABC reagent, Vector Laboratories), and diluted 1:250 in the same solution as the primary antiserum. The immunoreactivity was revealed with 3,3′-diaminobenzidine (DAB, Sigma-Aldrich Co., St. Louis, MO, USA) in 0.01 M PBS buffer and mounted on the gelatin-coated slides.

Immunocytochemistry was performed as we previously described^{39 (link)}. Briefly, cultured neurospheres or differentiated NPCs were washed with cold PBS (pH 7.4), and fixed with 4% paraformaldehyde (PFA) for 30 min. The cells were then washed with cold PBS containing 0.1% Tween for 4 times, 5 min each wash, and further blocked with 5% FBS for 1 h. Next the cells were incubated with the following primary antibodies: anti-Nestin (1:300, Abcam), anti-doublecortin (Dcx) (1:300, Abcam), mouse anti-BrdU (1:100; Roche Applied Science), mouse anti-β-tubulin III (Tuj-1, 1:500; Covance), rabbit anti-β-catenin (1:1000, Abcam), rabbit anti-glial fibrillary acidic protein (GFAP) (1:500; Invitrogen), mouse anti-PSA-NCAM (1:200, ThermoFisher). After PBS washing, the cells were then incubated with the primary antibodies listed above and with Cy3- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Vectashield (Vector Laboratory, Burlingame, CA) was used to coverslip the immunocytochemic slides. Immunostaining was analyzed using a fluorescence microscope (Olympus BX51). Quantification of neurite length in differentiated immature neurons (Tuj-1 positive) was performed using ImageJ-Neurite Tracer.

Primary antibodies: mouse anti-fibrillarin hybridoma supernatant, (^{51 (link)}, 1: 10), rabbit anti- γH2AX (Millipore anti-phospho H2AX, 1:500), rabbit anti-histone H3K27me3 (Upstate, 1:500), rabbit anti-histone H3K9me2 (Active Motif, 1:1000), mouse anti-5-Methylcytosine antibody (Epigentek, 1:100), human anti-topoisomerase I (^{52 (link)}, 1:100) and human anti-Pol I (SLR Research Corporation, 1:10). Secondary antibodies: anti-human FITC (Chemicon, 1:100), anti-mouse Alexa488 (Invitrogen, 1:1000), anti-rabbit Alexa488 (Invitrogen, 1:200), anti-mouse Rhodamine F(ab)2 (Chemicon, 1:10), anti-DIG Rhodamine Fab fragments (Roche, 1:200), mouse anti-BrdU (Roche, 1:15).