Mouse anti s100β

Coronal brain sections (30 μm thickness) isolated from mice 7 days following PT induction were prepared using a cryotome and collected in PBS. Free-floating sections were then permeabilized with PBS-0.25% Triton-X, blocked with 5% FBS, and treated with the following primary antibodies: Rabbit anti-GFAP (1:750; DAKO), goat anti-meteorin (1:100; R&D Systems, Minneapolis, MN, USA), and mouse anti-S100β (1:500; Sigma-Aldrich) in 5% FBS overnight at 4°C. Following extensive washing with PBS-0.25% Triton-X, the sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:100) for 2 h at room temperature, and the tissues were mounted using FluorSave™ Reagent (EMD Millipore, Billerica, MA, USA). Images of the tissues were captured using an M200 ApoTome microscope (Carl Zeiss AG, Oberkochen, Germany) and a LSM700 confocal microscope (Carl Zeiss AG).

Cultured astrocytes were fixed with 4% paraformaldehyde (PFA) for 10 min at 3DIV. Cells were blocked in 50% goat serum and immunostained with 1∶1000 rabbit-anti-GFAP (Dako Z0334) for 2 hours at room temperature followed by an Alexa594 conjugated secondary antibody. Other antibodies were used at the following concentrations: rabbit-anti-AQP4 (Sigma A5971) at 1∶1000, mouse-anti-S100β (Sigma S2532) at 1∶200, rabbit-anti-Sox2 (abcam 97959) at 1∶1000, mouse-anti-Nestin (BD Biosciences 556309) at 1∶1000, mouse-anti-HuC/HuD (Life Technologies A21271) at 1∶10, mouse-anti-Tuj1 (Sigma T8660) at 1∶1000, rabbit-anti-NG2 (Millipore AB5320B) at 1∶500 and rat-anti-MBP (abcam 7349) at 1∶300. For morphology analyses, cell membranes were then labeled by incubation with 1x HCS CellMask Green (Life Technologies H32714) in PBS for 30 min. Cells were mounted in DAPI Fluoromount-G (Southern Biotech) and duplicate wells from 3 independent experiments imaged at 10x on an inverted Zeiss microscope. Morphology and process length were determined from the CellMask membrane staining images and quantification done using the Sholl Analysis plugin in ImageJ [22] (link). Primary process number was calculated at a radius of 20 µm from the center of the cell body. Significance determined using one-way ANOVA with Tukey correction.

Antibodies used in this study are in the followings: mouse anti-E-cad (BD Transduction Laboratories 61081, 1:250); chick anti-GFAP (Millipore, 1:5,000); chick anti-GFP IgY (Abcam ab13970, 1:5,000); rabbit anti-GLAST polyclonal antibody (Covalab (France) PA-INS, 1:1,000); rabbit anti-Ki67 polyclonal antibody (Novocastra, NCL-Ki67p, 1:1,000); goat anti-NeuroD antibody (N-19; Santa Cruz, sc-1,084, 1:1,000); rabbit anti-Olig2 IgG (Millipore, AB9610, 1:1,000); goat anti-Olig2 (R&D systems, 1:1,000); mouse anti-PCNA (Novocastra, PC-10, 1:100); rabbit anti-PSmad3(S423/S425) polyclonal antibody (Millipore 07-1389, 1:1,000); mouse anti-phospho-vimentin IgG (MBL, D076-3S, 1:1,000); goat anti-Prox1 IgG (R&D systems, 1:1,000); mouse anti-S100β (Sigma, 1:1,000); rabbit Sox2 antibody (Millipore, 1:1,000); goat anti-Sox2 IgG (R&D systems, 1:1,000); goat anti-Sox9 polyclonal antibody (R&D systems, 1:1,000).

Cell monolayers and NSPs were fixed in 4% PFA for 20 minutes at 4°C and then washed briefly in PBS. NSPs were embedded in Tissue-Tek OCT compound (Labtek) and cut at 15 μm on a cryostat, and sections were placed on superfrost slides. After washing in PBS, sections or culture dishes were permeabilized with 0.1% Triton-X-100 in PBS (PBT) for 5 minutes and then blocked in 10% fetal calf serum in PBT for 60 minutes at room temperature. Samples were then incubated with primary antibody (diluted in the block buffer) overnight at 4°C. The following primary antibodies were used: goat anti-SOX2 (1 : 200, R&D Systems), mouse anti-PAX6 (1 : 80, DSHB), rabbit anti-TBR2 (1 : 1000, Chemicon), mouse anti-TUJ1 (1 : 1000, Millipore), rabbit anti-KI67 (1 : 600, Abcam), and mouse anti-S100β (1 : 500, Sigma). Following three 5 minute washes in PBT, ALEXA Fluor secondary antibodies (Life Technologies/Invitrogen) (1 : 1000 diluted in the block buffer) were applied for 1 hour at room temperature. All samples were counterstained with 49,6-diamidino-2-phenylindole (DAPI; 1 μg/ml, Sigma-Aldrich). Samples were then mounted onto glass slides with 5 μl of moviol aqueous mountant followed by viewing and image capturing under Zeiss Axio Observer z1 fluorescence microscope using ZEN imaging software.

Antibodies for immunostaining included chicken anti-GFP (1:1000 dilution, Abcam, Cat# ab13970), rat anti-GFP (1:1000, Nacalai Tesque, Cat# GF090R), anti-Sox2 (1:200, Cell Signaling Technology, Cat# 3728), anti-RFP (1:1000, MBL, Cat# PM005), anti-Fabp7 (BLBP, 1:500, Millipore, Cat# ABN14), mouse anti-S100β (1:200, Sigma-Aldrich, Cat# S2657), rabbit anti-S100β (1:500, Abcam, Cat# ab52642), anti-NICD1 (1:200, Cell Signaling Technology, Cat# 4147), anti-GFAP (1:1000, Abcam, Cat# ab4674), anti-Hey1 (1:200, Millipore, Cat# AB5714), anti-Ascl1 (1:200, BD Biosciences, Cat# 556604), anti-PCNA (1:500, Millipore, Cat# NA03), anti-Dcx (1:1000, Abcam, Cat# ab18723), anti-EGFR (1:500, Fitzgerald, Cat# 20-ES04), anti-Vcam1 (1:500, BD Biosciences, Cat# 550547), Hoechst 33342 (1:10000, Molecular Probes).

Animals were anesthetized using 1% pentobarbital sodium and then perfused transcardially with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde (PFA). Mouse brains were fixed with 4% paraformaldehyde solution for 6 h and were dehydrated in 30% sucrose for one day at 4 °C. Coronal brain sections (20 μm) were cut with a microtome (Leica), bonded on glass slides for immunostaining in choroid plexus, and stored in 0.1 M PBS for immunostaining in the hippocampus. The sections were incubated with primary antibody at 4 °C overnight. After washing with PBS three times, sections were incubated with the secondary antibody. The antibodies used were goat anti-TTR (1:50, LifeSpan, Cat: 395,788), rabbit anti-CAR1 (1:200, Proteintech, Cat:13198-2-AP), mouse anti-NeuN (1:500, Abcam, Cat: ab104224), goat anti-GFAP (1:1000, Abcam, Cat: ab53554), mouse anti-S100β (1:1000, Sigma, Cat:AMAB91038), goat anti-Iba1 (1:100, Abcam, Cat: ab5076), mouse anti-MBP (1:500, Abcam, Cat: ab254026), Alexa Fluor 488 donkey anti-rabbit IgG (Cat: R37118), Alexa Fluor 555 donkey anti-goat IgG (Cat: A-21,432), Alexa Fluor 594 donkey anti-mouse IgG (Cat: A-11,058) (all 1:1000 and all is from Invitrogen). All fluorescent images of the mounted sections were captured with a Nikon A1R confocal microscope.