Mouse anti active β catenin anti abc

Cultured cells were fixed with 4% PFA solution for 10 minutes and permeabilized in PBS with 0.3% Tween-20 for 10 minutes followed by blocking with PBS containing 1% bovine serum albumin (BSA) and 0.15% Tween-20 for 1 hour at room temperature. The cells were incubated with primary antibodies diluted in PBS (1:500) overnight at 4°C. Primary antibodies used were as follows: Rat anti-MBP (Millipore MAB386), rabbit anti-Olig2 (Millipore AB9610), guinea pig anti-Olig2 (from B. Novitch), mouse anti-MBP (Covance SMI-99P-100), mouse anti-beta-Tubulin III (Sigma-Aldrich T8578), Rabbit anti-Neurofilament H (Millipore AB1989), rabbit anti-GFAP (Agilent Z0334), mouse anti-Active-β-catenin (Anti-ABC) (Millipore 05–665). After incubation with appropriate secondary antibodies (1 hour room temperature), cells were counterstained with DAPI (Invitrogen R37606). To visualize F-actin network, cells were stained with Alexa Fluor 488-phalloidin (Invitrogen A12379) during secondary antibody incubation. Cells were mounted on slides with mounting reagent and imaged using confocal microscope (Zeiss LSM700) with 20x objective and on epifluorescence microscope (Keyence BZ-X710) with 10x objective for tiling (3×3).