Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0. 1% Triton X-100 permeabilized C2C12 cells labeling Nkx2-5 with BD-PB3237 at 1/25 dilution, followed by Dylight? 488-conjugated goat anti-Rabbit IgG secondary antibody at 1/200 dilution (green). Immunofluorescence image showing Nucleus staining on C2C12 cell line. Cytoplasmic actin is detected with Dylight? 554 Phalloidin(red). The nuclear counter stain is DAPI (blue).

Overlay histogram showing C2C12 cells stained with BD-PB3237(green line). The cells were fixed with 2% paraformaldehyde and then permeabilized with 90% methanol for 10 min. The cells were then incubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody (1:25 dilution) for 60 min at 37oC. The secondary antibody used was Goat-Anti-Rabbit IgG, DyLight? 488 Conjugated Highly Cross-Adsorbed at 1/200 dilution for 40 min at Room temperature. Isotype control antibody (blue line) was rabbit IgG1 (1μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

Anti-Mouse Nkx2-5 Antibody (Center) at 1:1000 dilution + CCRF-CEM whole cell lysate Lysates/proteins at 20 μg per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size : 30-42 kDa Blocking/Dilution buffer: 5% NFDM/TBST.