de novo methylation. Methylation of CpG-dinucleotides was detected using a rabbit polyclonal antibody against methyl-CpG binding domain 1 (MBD1, 1:200, Millipore). AZA treated cultures were processed for immunocytochemical staining to assess DNA methylation (DNMT1a and 3a; 5-MeC antibodies) and histone marks (2me-H3K4, 3me-H3K27 antibodies) (n= 4-5 of 14 /staining; 2 control with pre-immune serum) and was confirmed in triplicate. The above antibodies were commercially characterized for their titer as well as specificity, and screened through our initial staining in vivo with embryonic tissue for confirmation of their intranuclear staining and known distribution. There were 4-5 wells for each immunostaining containing 2-3 neurospheres per well; neurospheres were sampled from each of the 4 wells for analysis.
