Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/2000 dilution (purified) + SW480 cell lysate at 20 µg

Secondary
Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 53 kDa
Observed band size: 53 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling HNF-4-alpha with purified ab92378 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling HNF-4 with purified ab92378 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/5000 dilution (purified) + HepG2 cell lysate at 10 µg

Secondary
Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 53 kDa
Observed band size: 53 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 cell lysate
Lane 2 : A549 cell lysate
Lane 3 : SW480 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 53 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue (A) and human kidney tissue (B) labelling HNF-4-aplha with unpurified ab92378 at a 1/100 dilution. Detection: DAB staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunocytochemistry/Immunfluorescence analysis of HepG2 cells labelling HNF-4-alpha with unpurified ab92378 at a 1/100 dilution.

Flow Cytometry analysis of HepG2 cells labelling HNF-4 with purified ab92378 at 1/70 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Overlay histogram showing HepG2 cells stained with unpurified ab92378 (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 (unpurified ab92378, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/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 with 0.1% PBS-Tween for 20 min used under the same conditions.