Anti-Cathepsin K antibody [3F9] (ab37259) at 1 µg/ml + Bone (Human) Tissue Lysate - tumor tissue (ab29359) at 10 µgSecondaryGoat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/3000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.

Overlay histogram showing U20S cells stained with ab37259 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab37259, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in U20S cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.