Staining of PAX9 protein in human oesophageal epithelium. Magnification x200

All lanes : Anti-PAX9 antibody [7C2] (ab28538)

Lane 1 : mouse oesophageal protein extract
Lane 2 : human oesophageal protein extract

Predicted band size: 38 kDa
Observed band size: 38 kDa

ICC/IF image of ab28538 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab28538, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rat IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing HepG2 cells stained with ab28538 (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 followed by the antibody (ab28538, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was goat anti-rat DyLight® 488  (IgG H+L) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG1, kappa monoclonal [RTK2071] (ab18412, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.