Rabbit anti foxm1

Sections of paraffin-embedded neutralized buffered formalin fixed tissue were used for Ni-DAB (FoxM1) or DAB (other antibodies) colorimetric immunohistochemistry (IHC), while paraformaldehyde fixed frozen tissue sections or cells grown on coverslips were subjected to immunofluorescence (IF). Antibodies include rabbit anti-FoxM1 (1:1000 for IHC or 1:500 for IF; Santa Cruz, cat# sc-502), rabbit anti-cyclin D3 (1:500; Santa Cruz), rabbit anti-Ki67 (1:500; Thermo), rat anti-BrdU (1:200; Abcam), rat anti-phosphorylated histone H3 (Ser 10) (1: 1000; Millipore), rabbit anti-PR (1:300; Santa Cruz), rabbit anti-ERα (1:300; Santa Cruz), and rabbit anti-cleaved caspase 3 (1:300 for IF; Santa Cruz). Counting of immunostaining-positive cells were determined using the Image J program available at http://imagej.nih.gov/ij (NIH, USA), and the analyses were based on examination of serial sections for at least 5–6 IS samples collected from 3–5 different mice for each group.

Immunofluorescence experiments were performed using poly-lysine coated Labtek chamber slides as support: NSCs were allowed to adhere for three hours, while for differentiation studies, NSCs were dissociated and cultured as described in “Murine cerebellar NSC cultures”. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and incubated in blocking solution (5% normal goat serum (NGS), 1% BSA, 0.1% Triton X-100). Cells were incubated overnight with primary antibodies diluted in blocking solution and for 2 h with secondary antibodies. Primary antibodies were rabbit anti-Nanog polyclonal (Abcam ab80892); mouse anti-Nestin (Abcamab 11306); mouse anti-Gli1, #2643 (Cell Signaling Technology Inc); mouse anti-parvalbumin P3088 (Sigma); rabbit anti-S100 S2644 (Sigma); mouse anti-βIII-tubulin (βIIItub) MAB1637 (Millipore); rabbit anti-Foxm1 (Santa Cruz Biotechnology, sc-502). Secondary antibodies (488-conjugated anti-mouse and anti-rabbit) were purchased from Molecular Probes (Invitrogen). Nuclei were Hoechst-counterstained and cover slips were mounted with fluorescence mounting medium (S3023) (Dako). Images were acquired with a Carl Zeiss microscope (Axio Observer Z1) using Apotome technology and AxioVision Digital Image Processing Software.

Cells were lysed in Tris–HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM, and protease inhibitors. Lysates were separated on an 8% acrylamide gel and immunoblotted using standard procedures. Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk and incubated overnight at 4 °C with the following antibodies: rabbit anti-Foxm1 (Santa Cruz Biotechnology, sc-502), rabbit anti-Hsp70, (Santa Cruz Biotechnology, sc-33575), mouse anti- βIIItub (MAB 1637 Millipore), goat anti-actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-Nanog (PA1–41577, Thermo Fisher), anti-α-Tubulin Antibody (T9026 SIGMA). HRP-conjugated secondary antisera (Santa Cruz Biotechnology) were applied and binding visualized by enhanced chemiluminescence (ECL Amersham).