Ab41548

Immunoprecipitation analysis of macrophages was performed as described^{20 (link)}. Conditions were as for in situ proximity ligation assay except that isolated macrophages, following treatment for 30 min with 200 ng/ml S100B or vehicle, were subjected to immunoprecipitation with anti-S100B antibody (Abcam No. ab41548; 1 µg/ml). Immunoprecipitates were then probed with anti-S100B and anti-FGFR1 antibodies.

Sections (6 μm) were prepared from formalin-fixed paraffin-embedded blocks for IHC stains with adequate controls. IHC was performed using the Avidin/Biotin blocking kit (Vector Labs, SP-2001) staining with antibodies against S100 (1:250 dilution; catalog ab41548, Abcam), KI-67 (prediluted; catalog 790-4286, Ventana), and COL4A (1:1000 dilution; catalog ab6586, Abcam) as previously described (49 (link)). Immunolabeling for anti-histone H3 (tri methyl K27) (1:200 dilution; catalog ab6002, Abcam) was performed on formalin-fixed, paraffin embedded sections. Briefly, following dewaxing and rehydration, slides were immersed in 1% tween-20; then, heat-induced antigen retrieval was performed in a steamer using 1.0 mM EDTA pH 9.0 (catalog AP9004500, Thermo Fisher Scientific) for 45 minutes. Slides were rinsed in PBST, and endogenous peroxidase and phosphatase was blocked (catalog S2003, Dako). Sections were then incubated with primary antibody anti–histone H3 (tri methyl K27) (1:200 dilution; catalog ab6002, Abcam) for overnight at 4°C. The primary antibodies were detected by 30-minute incubation with HRP-labeled secondary antibody (catalog PV6114, Leica Microsystems), followed by detection with 3,3′-Diaminobenzidine (Sigma-Aldrich), counterstaining with Harris hematoxylin, dehydration, and mounting.

After 3 weeks (Control and Runner groups) or 6 weeks (Runner-Rest group), mice were perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Brains were removed and post-fixed in 4% PFA. Coronal sections were cut at 40-μm thickness on a vibratome (Dosaka EM, Japan).
Immunohistochemistry was performed as described previously (Tatsumi et al., 2008 (link)) The primary antibodies were used at the following dilutions: anti-GFP (1:10,000, rabbit polyclonal, A6455, Thermo Fisher Scientific; 1:2000, rat monoclonal IgG2a, clone GF090R, Nacalai Tesque, Japan), anti-NG2 proteoglycan (1:200, rabbit polyclonal, AB5320, EMD Millipore, USA), anti-adenomatous polyposis coli protein (APC) (1:500, mouse monoclonal, clone CC-1, EMD Millipore), anti- S100 calcium-binding protein B(S100β) (1:5000, rabbit polyclonal, AB41548, Abcam, Japan), anti-GABA transporter 3 (1:200, rabbit polyclonal, AB1574, EMD Millipore). Alexa conjugated antibodies (1:1000, Jackson ImmunoResearch Laboratories, UK), or biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, USA) were used as secondary antibodies.
To minimize artifactual variation in fluorescence intensity across animals and groups, GFP immunofluorescence procedures for all sections of three groups were performed simultaneously under the same staining conditions.

Slide-mounted sections were warmed (37°C, 5 min), equilibrated in PBS (5 min, RT), fixed in PFA (4% in PBS; 10 min, RT), washed in PBS (3 min, RT), permeabilized with Triton X-100 (0.5% in PBS; 30 min, RT), washed in TNT (3 × 5 min, RT), blocked in fetal bovine serum (FBS; 10% in TN; 30 min, RT), incubated with mouse anti-NEUN (EMD Millipore, Billerica, MA, MAB377; 1:1000) and rabbit anti-S100ß (Abcam, UK, ab41548; 1:500) antibodies (in 10% FBS; 12 hr, 4°C), washed in TNT (3 × 5 min, RT), incubated with secondary antibodies (Alexa488- and Alexa647-labeled; Invitrogen, Carlsbad, CA; 1:1000 in 10% FBS; 12 hr, 4°C), and washed in TNT (3 × 15 min, RT). Slides were mounted using Vectashield containing DAPI (5 μg/ml).

Cells were fixed with 4% paraformaldehyde for 20 min and washed. Unspecific antibody binding was blocked using 5% normal goat serum followed by incubation with primary antibodies overnight at 4 °C. After washing, the cells were incubated with their respective Alexa conjugated secondary antibodies (Thermo-Scientific, Bratislava, Slovakia) for 1 h at room temperature in dark. The slides were mounted with coverslips using Fluorshield^TM with DAPI to visualize nuclei (Sigma). The following antibodies were used: GFAP (1:500, ab10062; Abcam, Bratislava, Slovakia), ACSA-1 (GLAST) (1:500; #130-095-822; Miltenyi biotech), ALDH1L1 (1:500; MABN495; Bratislava, Millipore), S100β (1:500; ab41548; Abcam), β-tubulin III (Tuj1) (1:500; ab78078; Abcam), MAP-2 (1:500; ab5392; Abcam), Synapsin 1 (1:1000; ab64581; Abcam), PAX-6 (1:1000; ab195045; Abcam), SOX-2 (1:500; ab92494; Abcam), and Nestin (1:500; ab22035; Abcam).