Alexa flour 488 labeled anti mouse igg

Osteosarcoma and osteoblast cells, grown on cover slips, were treated with either DMSO or 25 μmol/l AR-A014418 for 24 hrs and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X (Sigma-Aldrich). The fixed and permeabilized cells were incubated with mouse monoclonal antibody to β-catenin (BD Bioscience; diluted 1:200) at 4°C overnight and then with Alexa Flour^® 488-labeled anti-mouse IgG (Invitrogen; diluted 1:1,000) at room temperature for 40 min. After washing off excess antibody, cell nuclei were counterstained with Hoechst 33342 (Molecular Probes) for 20 min at room temperature. The cells were observed by fluorescence microscopy (Keyence) for the expression and subcellular localization of β-catenin. The effect of GSK-3β inhibition on the subcellular localization of β-catenin was quantified in cells treated with DMSO or with AR-A014418. In each assay, the mean percentage of cells that was positive for nuclear β-catenin localization was calculated with SDs in 3 separate microscopic fields.

Immunohistochemistry was performed as previously described with minor modifications [8 ]. After deparaffinization, rehydration, and antigen retrieval (microwaved in 10 mM citrate buffer, pH 6.0, at 100 °C, 5 min, three times), the sections were incubated sequentially with 0.5% TritonX-100 in PBS for 30 min at room temperature (RT) to membrane permeation, with 10% FBS for 60 min at RT, with primary antibodies: rabbit anti-phospho-LATS1 (Ser909, 1:100, Cell Signaling Technology, MA, USA, #9157), rabbit anti-phospho-PLK1 (Thr210, 1:100, Abcam, Cambridge, UK, #ab155095), mouse anti-MAP2 (1:200, Santa Cruz, TX, USA, #sc-32791) and mouse anti-NeuN (1:100, Abcam, Cambridge, UK, ab104224) one or two overnight, and finally with secondary antibodies: Alexa Flour 488-labeled anti-mouse IgG (1:1000, Invitrogen, MA, USA) and Cy3-labeled anti-rabbit IgG (1:500, Jackson ImmunoResearch, PA, USA) for 1 h at RT. Images were acquired by confocal microscopy: Olympus FV1200 (Olympus, Tokyo, Japan) and LSM710 (Carl Zeiss, Oberkochen, Germany).