Brains were perfusion-fixed with 4% paraformaldehyde and postfixed in the same fixative overnight, and 50 μm sections were cut on a Vibratome sectioning system (VT1000S; Leica, Heidelberg, Germany, http://www.leica.com). After three rinses in PBS, some sections were incubated either in acetone for 20 seconds on ice (for staining β-catenin) or in 2 N HCl for 30 minutes (for staining BrdU, proliferating cell nuclear antigen [PCNA], and Mash1). The sections were then incubated for 1 hour in TNB blocking solution (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), overnight with primary antibodies, followed by a 60-minute incubation at room temperature with biotinylated secondary antibodies (1:200) or Alexa Fluor-conjugated secondary antibodies (1:200; Molecular Probes Inc., Eugene, OR, http://www.probes.invitrogen.com), unless otherwise noted. Biotinylated antibodies were visualized using the ABC Elite kit (Vector Laboratories) and TSA (PerkinElmer Life and Analytical Sciences). The primary antibodies (final dilution and source) used in this study were as follows: mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:100) (catalog number G3893; Sigma-Aldrich), rabbit polyclonal anti-GFAP (1:1,000) (catalog number Z0334; Dako, Glostrup, Denmark, http://www.dako.com), mouse monoclonal anti-PSA-NCAM IgM (1:250, a gift from Tatsunori Seki) , mouse monoclonal anti-Mash1 (1:100) (catalog number 556604; BD Pharmingen), rat monoclonal anti-BrdU (1:100) (catalog number ab6326; Abcam, Cambridge, U.K., http://www.abcam.com), rabbit anti-GFP (1:200) (catalog number 598; MBL International Corp., Woburn, MA, http://www.mblintl.com), goat anti-doublecortin (anti-DCX) (1:200) (catalog number sc-8066; Santa Cruz Biotechnology), anti-phosphohistone H3 (1:200) (catalog number 06-570; Upstate), anti-β-catenin (1:200) (catalog number 610154; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), anti-PCNA (1:200) (catalog number NAO3T; Siemens Medical Solutions Diagnostics, Oncogene Science Biomarker Group, Cambridge, MA, http://www.oncogene.com), anti-NeuN (1:200) (catalog number MAB377; Chemicon, Temecula, CA, http://www.chemicon.com), and mouse monoclonal anti-cleaved Caspase-3 (1:200) (catalog number 9661; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com).
These antibodies have been shown to be specific in the following ways. The anti-GFP and anti-BrdU antibodies used in this study did not show any staining in brain sections of normal, untreated mice. The antibodies against anti-GFAP , PSA-NCAM , Mash1 , DCX , phosphohistone H3 , PCNA , NeuN , and cleaved Caspase-3  have been widely used as specific markers for the immunohistochemistry of brain sections. The labeling patterns we obtained with these antibodies were consistent with previous reports. The anti-β-catenin antibody used in this study was purchased from Becton Dickinson and stains a single band of 92 kDa on Western blots (according to the manufacturer's technical information). This antibody also produced a staining pattern similar to that previously reported . The number of SVZ cells containing nuclear β-catenin, which were detected using this antibody, was increased by GSK3β inhibitor injection, as expected for the specific labeling of β-catenin (Fig. 3). Finally, we further confirmed the specificity of this antibody by staining brain sections of mice that had received injections of pMX-IRES-GFP or pMX-ΔN90β-catenin-IRES-GFP retrovirus into the SVZ. The β-catenin signal was clearly colocalized with GFP in the SVZ treated with pMX-ΔN90β-catenin-IRES-GFP but not that treated with pMX-IRES-GFP (supplemental online Fig. 1), indicating that the signal produced by this antibody represents specific immunoreactivity with the β-catenin protein.