Ab70493

Mouse ES cells were washed with PBS, fixed in 3.7% formaldehyde in PBS for 30 min at r.t., and then permeabilized with 0.5% Triton X-100 in PBS for 5 min at r.t. The cells were blocked with 5% FCS in PBS for 1 h at r.t., and incubated with primary antibodies. After washing 3 times with 5% FCS in PBS, the cells were incubated with fluorescently labeled secondary antibodies for 1 h at r.t., and cell nuclei were stained with 4 " ,6 -diamidino -2-phenylindole (Vector Laboratories, Burlingame, CA, USA). The cells were observed under a fluorescence microscope (IX70 ; Olympus, Tokyo, Japan) equipped with CoolSNAP HQ2 (Photometrics, Tucson, AZ, USA) and images were processed using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). A confocal fluorescence microscope (FV1000 ; Olympus) was also used to analyze the cells. The antibodies used for the immunofluorescent staining were as follows : FBL (ab4566 ; abcam) and Nucleolin (ab70493 ; abcam).