ab212096 staining Smad2 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab212096 at 1/100 dilution (pseudocolored in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Overlay histogram showing MCF7 cells stained with ab212096 (red line). The cells were fixed with 4% formaldehyde and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab212096, 1/1000 dilution) for 30 min at 22°C.          

Isotype control antibody (black line) was rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 50mW Yellow/Green laser (561nm) and 586/15 bandpass filter.               

This antibody gave a positive signal in MCF7 cells fixed with 4% formaldehyde , permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.