Immunocytochemistry/Immunofluorescence analysis of alpha 1 Sodium Potassium ATPase shows staining in MCF-7 cells. alpha 1 Sodium Potassium ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2867 (1:20) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of alpha 1 Sodium Potassium ATPase shows staining in NIH-3T3 cells. alpha 1 Sodium Potassium ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2867 (1:20) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

Immunohistochemistry (Formail/PFA-fixed paraffin-embedded sections) was performed on human brain tissue. Antigen retrieval was performed using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab2867 (1:10) overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Immunohistochemistry (Formail/PFA-fixed paraffin-embedded sections) was performed on human liver tissue. Antigen retrieval was performed using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab2867 (1:10) overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Immunohistochemistry (Formail/PFA-fixed paraffin-embedded sections) was performed on human renal carcinoma tissue. Antigen retrieval was performed using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab2867 (1:10) overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Overlay histogram showing HEK293 cells stained with ab2867 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab2867, 1/50 dilution) 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 IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

ICC/IF image of ab2867 stained HepG2 cells. The cells were 4% formaldehyde 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 (ab2867, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 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.