Sc 359850

MG63 cells were washed three times with PBS and fixed with Immunol Staining Fix Solution (Beyotime) for 10 min, after which time they were washed with Immunol Staining Wash Buffer (Beyotime) 3 times for 5 min each. Actin-Tracker Green was diluted in Immunol Fluorescence Staining Secondary Antibody Dilution Buffer (Beyotime) and incubated with the cells for 40 min in the dark, after which they were washed with Immunol Staining Wash Buffer 3 times for 5 min each. Finally, nuclei were stained with UltraCruz^® Hard-set Mounting Medium including 4′,6-diamidino-2-phenylindole (DAPI; sc-359850, Santa Cruz Biotechnology). Images were recorded using a Zeiss confocal laser-scanning microscope equipped with a Plan-Apochromat 60× oil-immersion objective lens (Zeiss LSM 880 Confocal Laser Scanning unit, Carl Zeiss AG, Oberkochen, Germany). Image processing, combining and analysis were performed using the ZEN software package (Carl Zeiss MicroImaging GmbH, Jena, Germany).

Transfected cells were grown on 0.1% gelatin-coated glass chip in 24-well plates. Cells were fixed in 4% paraformaldehyde 24 h after initial plating, permeabilized with 2% NP-40, then blocked and incubated overnight with 10% FBS in PBS containing primary antibodies. Primary antibodies were visualized with Alexa-350 (anti-mouse from Life Technologies), Alexa-488 (anti-rabbit, anti-mouse, and anti-goat from Invitrogen) and Alexa-594 (anti-rabbit, anti-mouse, and anti-rat from Invitrogen) secondary antibodies. Nuclei were visualized with 2 μg/ml Hoechst. Slides were mounted with Dako Fluorescent Mounting Medium (Dako) or UltraCruz Hard-set Mounting Medium containing 1.5 μg/ml DAPI (sc-359850, Santa Cruz Biotechnology) and visualized by fluorescence microscopy (Axioplan2, Zeiss; Olympus IX71, Olympus).