Immunofluorescence localization of β-catenin

MC3T3-E1 cells were plated at a density of 1.5 × 10⁴ cells/cm² on 12-mm glass coverslips in Falcon 24-well plates in culture medium. After 2 days, cells were placed in serum-free medium and incubated overnight. On the day of the experiment, cells were incubated with test substances for the indicated times. Cells were then fixed with paraformaldehyde (4 %) in sucrose solution (2 %), permeabilized with 0.1 % Triton X-100 in DPBS for 10 min, and blocked for 1 h with 1 % normal goat serum in DPBS (blocking solution). To detect subcellular localization of β-catenin, cells were first incubated for 1 h with a mouse monoclonal antibody (1:200 in blocking solution) followed by 2-h incubation with an Alexa Fluor® 488 goat anti-mouse antibody (1:200 in blocking solution) at room temperature. Stained samples were then sealed using Vectashield mounting medium with DAPI and visualized by confocal microscopy (model LSM 510; Carl Zeiss Inc., Jena, Germany) using a Zeiss Plan-Apochromat 40× objective (1.2 NA) at a slice thickness of 2 μm with 488-nm Ar⁺ ion laser excitation and emission wavelengths filtered at 500–550 nm band pass. To quantify subcellular localization of β-catenin, the average fluorescence intensity of an area in the nucleus (F_N) and the average fluorescence intensity of an area of equal size in the cytosol (F_C) were determined. Values of the ratio F_N/F_C greater than or equal to 1.25 were taken to indicate nuclear localization of β-catenin protein. Twelve images were obtained per coverslip for each treatment, and the F_N/F_C ratios for all cells within a field were analyzed (∼20–30 cells per field).