Immunohistochemistry for Protein Localization

Immunohistochemistry was performed as previously described^{54 (link)}. 3,3-Diaminobenzidine (Sigma) stains were performed using an ABC labeling kit (Vector Laboratories, Burlingame, CA). Primary antibodies were chosen according to previous studies in our lab or reports in the literature or instructions from the vendors: Rabbit anti-P-Smad2 (1:1000; Chemicon, AB3849), rabbit anti-eGFP (1:500; Life Technologies, A11122), chicken anti-eGFP (1:1000, Avis, GFP-1020), mouse anti-mCherry (1:200, Clontech, 632543), chicken anti-Tbr2 (1:500, Millipore, AB15894), mouse anti-MCM2 (1:500, BD Biosciences, 610700), rat anti-BrdU (1:1000; Abcam, AB6326), goat anti-DCX (1:500; Santa Cruz Biotechnology, sc-8066), mouse anti-PCNA (1:200; DAKO, M0879), rabbit anti-GFAP (1:1000; DAKO, Z0334), mouse anti-NeuN (1:1000; Millipore, MAB377), goat anti-Sox2 (1:200, Santa Cluz Biotechnology, sc-17320), mouse anti-HA (1:1000; Covance, MMS-101P), rabbit anti-c-fos (1:10000; Millipore, PC38). Antigen retrieval with 3 mol/L HCl was used for BrdU. For fluorescent stains, secondary antibodies were purchased from either Molecular Probes or Jackson Immunoresearch. For fluorescent staining procedures, final washes included the nuclear stain Topro-3 (1:500; Molecular Probes) or DAPI (5 μg/mL, Sigma) for 0.5 h at room temperature.