Recombinant Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Human kidney tissue using unpurified ab109225.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ICC/IF image of unpurified ab109255 stained HeLa 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 (ab109225, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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.

Immunohistochemical staining of paraffin embedded human stomach with purified ab109225 at a dilution of 1/500. A HRP goat anti-rabbit (ab97051) was used as the secondary antibody at a dilution of 1/500 and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescent staining of HeLa cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab109225 at a dilution of 1/200. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counter stained with DAPI. The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

Intracellular Flow Cytometry analysis of U87-MG (human glioblastoma) cells labeling NADPH oxidase 4 with purified ab109225 at 1/280 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab109225 (purified) at 1/30 immunoprecipitating NADPH oxidase 4 in HEK293. For western blotting, a HRP-conjugated anti-rabbit antibody was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Unpurified ab109225 staining Nox4 in HeLa cells treated with (-)-cannabidiol (ab120448), by ICC/IF. Increase in Nox4 expression correlates with increased concentration of (-)-cannabidiol, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120448 ((-)-cannabidio) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab109225 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.