Anti actinin 4 antibody

Ros 17/2.8 cells and MG63 cells were plated on chamber slides (NalgeNunc International, Naperville, IL, USA). When the cells were extended thoroughly, they were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 25 min. Following three washes with PBS, the cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Then they were blocked with 1% BSA for 1 h at room temperature followed by three washes with PBS. The cells were incubated with anti-actinin 4 antibody (Abcam, Cambridge, MA, USA) and anti-β₃ subunit antibody (Alomone, Jerusalem, Israel) at 4°C over night and 30 min at room temperature the next day with gently rocking. After washing with PBS for 10 min for three times, the cells were incubated with the secondary antibodies with labeled with Rhodamine or FITC (Jackson Lab., Barharbor, Maine, USA) at room temperature for 2.5 h shield from light. After three washes with PBS likewise, cells were counterstained with DAPI nuclear stain for 5 min and washed once with PBS, and mounted with Vectashield and reserved at 4°C shield from light. The fluorescent signals were visualized by confocal microscopy (LSM 410; Carl Zeiss, Oberkochen, Germany).The co-localization of actinin 4 and L-type calcium channel β₃ subunit was determined by evaluating at least five samples.