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.