Abn14

Brains were processed through perfusion/fixation, as above. Serial 30 μm, free-floating coronal sections representing 1/6th of each brain were permeabilized in Tris-buffered saline containing Triton X-100 (TBST; 50 mM Tris with 0.9% NaCl, and 0.5% Triton X-100), incubated with 0.3% H₂O₂, and blocked with 10% horse serum/TBST. Primary antibodies were incubated in 1% horse serum/TBST for 48 h. Primary antibodies: glial fibrillary acidic protein (GFAP; 1:500, anti-rabbit, Dako Z033429), sex-determining region box 2 (Sox2; 1:100, anti-goat, Santa Cruz Biotechnology, sc-17320), Ki67 (1:500, Abcam ab15580), brain lipid binding protein (BLBP; 1:300, EMD Millipore, ABN14), doublecortin (DCX; 1:50, Santa Cruz Biotechnology, sc-8066, or 1:200 Abcam ab18723), FGF-23 (1:30, R&D Systems, MAB26291; 1:100, Mybiosource MBS2003657), cleaved caspase 3 (1:1000, Cell Signaling 9664), or S100β (1:400, Dako, Z0311). Primary antibody binding was visualized after incubation in fluorescently labeled secondary antibody (Life Technologies). Nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI; Life Technologies) and mounted in Prolong Gold anti-fade mounting media (Life Technologies).

Whole brains were fixed overnight in 4% paraformaldehyde in PBS and kept in 100% methanol at −20°C. Following rehydration, brains were either embedded in 3% agarose blocks and vibratome sectioned (50 µm) or processed for whole-mount immunohistochemistry. An antigen-retrieval step was performed for BrdU and/or Pcna immunolabelling: for BrdU, sections were incubated in 2 M HCl at room temperature for 30 min; for Pcna, brains were incubated in Histo-VT One (Nacalai Tesque) for 60 min at 65°C. The following primary antibodies were used: MCM5 (1:500, kindly provided by Soojin Ryu, Max Planck Institute for Medical Research, Heidelberg, Germany), anti-BLBP (1:1000, rabbit, Millipore, ABN14), anti-PCNA (1:250, mouse IgG2a, Santa Cruz Biotechnology, sc56; 1:500, rabbit, Genetex, GTX124496), anti-BrdU (1:150, rat IgG1, Abcam, ab6326), anti-GFP (1:1000, chicken, Aves laboratories, GFP 1020), anti-glutamine synthetase (1:1000, mouse IgG2a, Millipore, MAB302), anti-dsRed (1:250, rabbit, Clontech, 632496), anti-Sox2 (1:500, rabbit, Abcam, ab97959; 1:200, mouse IgG1, Abcam, ab171380) and anti-active Caspase-3 (1:300, rabbit, BD Pharmigen, 559565). Secondary antibodies raised in goat coupled to AlexaFluor dyes (Invitrogen) were used (1:1000).