Mouse monoclonal anti map2 antibody

Cells grown on glass coverslips were washed with 1x phosphate buffered saline (PBS); and fixed with pre-chilled methanol: acetone (1:1 v/v) for 5–10 min at room temperature. Fixed cells were permeabilized with 0.32% Triton X-100 in PBS for 15 min, and blocked with 5% goat serum in 1xPBS for 1 h. Cells were incubated with mouse monoclonal anti-MAP2 antibody (Sigma) at 4 °C for 24 h, washed thrice with 0.1% Triton X-100 in 1xPBS (PBST) for 5 min each and incubated with fluorescein isothiocyanate (FITC) conjugated goat anti mouse secondary antibody (Sigma). After three washings with PBST for 5 min each, coverslips were mounted on glass slides using 4′,6-diamidino-2-phenylindole (DAPI) mounting medium. Cells were visualized by Leica inverted fluorescence microscope (Leica DFC 450C).

Cells were fixed with Mildform 20N (8% formaldehyde; Wako) for 30 min and permeabilized with 0.2% Triton X-100 (nacalaitesque) for 5 min. Fixed cells were blocked with Blocking One solution (nacalaitesque) for 1h. The primary antibodies used here were: rabbit anti-human Nestin (1:200; N-1602; IBL Ltd.), mouse anti-neuron specific βIII-tubulin antibody (1:500; TuJ-1; R&D Systems), mouse monoclonal anti-MAP2 antibody (1:500; Sigma-Aldrich) and anti-mouse β-catenin (1:1000; BD Bioscience, USA). The samples were washed for three times, and incubated with goat anti-mouse IgG F(ab’)2-TRITC (1: 100; Santa Cruz) and anti-rabbit IgG F(ab’)2 Alexa Fluor 555 conjugate (1: 1000; Cell Signaling Technology) at room temperature for 1 h. After three times of washing, the samples were counterstained with 4’-6-diamidino-2-phenylindol (DAPI, Invitrogen), and examined by confocal inverted microscope (Nikon Eclipse Ti, Japan). Fluorescent images of approximately 400 cells in at least 3 different areas were analyzed for Tuj-positive cells. DAPI was used to quantify the total number of cells.