Immunocytochemistry analysis of PC-12 cells labelling nNOS (neuronal) (green) with purified ab76067 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.  Cells were counterstained with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red). Nuclear DNA was labelled with DAPI (blue).

Anti-nNOS (neuronal) antibody [EP1855Y] (ab76067) at 1/100 dilution (unpurified) + Mouse brain tissue lysate at 10 µg

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

Predicted band size: 161 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-nNOS (neuronal) antibody [EP1855Y] (ab76067) at 1/3000 dilution (purified) + Mouse brain tissue lysate at 10 µg

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

Predicted band size: 161 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-nNOS (neuronal) antibody [EP1855Y] (ab76067) at 1/1000 dilution (unpurified) + Mouse brain tissue lysate at 10 µg

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

Predicted band size: 161 kDa
Observed band size: 161 kDa

Unpurified ab76067 staining nNOS (neuronal) in murine sperm cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde and blocked using 2% BSA. Samples were then incubated with undiluted ab76067. The secondary used was a FITC conjugated goat anti-rabbit IgG at a 1/400 dilution.Panel A shows the specific staining of nNOS in sperm while Panel B is the control sample treated with Rabbit IgG.

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Overlay histogram showing PC-12 cells stained with unpurified ab76067 (red line). The cells were fixed with methanol (5 min) and 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 ab76067, 1/50 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 monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in PC-12 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.
Please note that Abcam do not have any data for use of this antibody in non-fixed cells. We welcome any customer feedback.

Flow cytometry analysis of PC-12 cells labelling nNos (neuronal) with unpurified ab76067 at 1/15 (red). Cells were fixed with 100% methanol. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

Flow cytometry analysis of PC-12 cells labelling nNos (neuronal) with purified ab76067 at 1/600 (red). Cells were fixed with 100% methanol. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

ab76067 (unpurified) at 1/4 immunoprecipitating nNOS (neuronal) in rat brain tissue lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

ab76067 (purified) at 1/150 immunoprecipitating nNOS (neuronal) in rat brain tissue lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.