All lanes : Anti-Aquaporin 3 antibody (ab125219) at 0.5 µg/ml (overnight at 4°C)

Lane 1 : Rat kidney tissue lysate
Lane 2 : Mouse kidney tissue lysate
Lane 3 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : COS-7 (African green monkey kidney fibroblast-like cell line) whole cell lysate

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : Goat Anti-rabbit IgG (HRP) for 1.5 hour at room temperature at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 32 kDa
Observed band size: 32 kDa
Additional bands at: 72 kDa. We are unsure as to the identity of these extra bands.

Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. The membrane was then blocked with 5% Non-fat Milk / TBS for 1.5 hours at room temperature followed by incubation with ab125219. The membrane was then washed with TBS-0.1% Tween 3 times for 5 minutes each and probed with a Goat anti-rabbit IgG (HRP) secondary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung cancer tissue labeling Aquaporin 3 with ab125219 at 1 μg/mL. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with ab125219 overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labeling Aquaporin 3 with ab125219 at 1 μg/mL. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with ab125219 overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labeling Aquaporin 3 with ab125219 at 1 μg/mL. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with ab125219 overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Immunocytochemistry analysis of A431 (human epidermoid carcinoma cell line) cells labeling Aquaporin 3 with ab125219 at 2 μg/mL. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum and then incubated with ab125219 overnight at 4°C. DyLight^® 488 Conjugated Goat Anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI and visualized using a fluorescence microscope with filter sets appropriate for the label used.

Overlay histogram showing A431 (human epidermoid carcinoma cell line) cells stained with ab125219 (Blue line). The cells were blocked with 10% normal goat serum and then incubated with rabbit anti-Aquaporin 3 antibody (ab125219, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight^® 488 conjugated goat anti-rabbit IgG (5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.