Anti-iNOS antibody (ab15323)

Apigenin reduced SP and iNOS expression in colonic myenteric ganglia from HFD mice.

Representative pictures of SP (substance P) and iNOS immunostaining of cross-sections from full-thickness mice colonic specimens from SD, SD plus treatment with apigenin (10 mg/Kg/day), HFD or HFD plus treatment with apigenin (10 mg/Kg/day). Scale bar = 20 μm.

Paraffin-sections were processed for immunoperoxidase staining. After an overnight incubation with primary antibodies against SP (code n. Sc-21715, Santa Cruz Biotech, California, USA) and iNOS (code n. ab 15323, Abcam, Cambridge, UK), the slices were exposed to appropriate biotinylated immunoglobulins, peroxidase-labelled streptavidin complex, and 3.3’-diaminobenzidine tetrahydrochloride (DakoCytomation, Glostrup, Denmark). The quantitative assessment of the immunostained sections was performed by the L.A.S. software v.4.5 Image Analysis System. The expression of antigens was evaluated as percentage of positive pixels (PPP) estimated over the total area of myenteric ganglia examined.

All lanes : Anti-iNOS antibody (ab15323) at 1 µg/ml

Lane 1 : Raw264.7
Lane 2 : Raw264.7 + LPS (1 ug per mL; 18 hours)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : IR- goat anti-rabbit (green) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 131 kDa

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system under denaturing, reducing conditions. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with rabbit polyclonal to iNOS (ab15323; 1 ug x mL-1 (based on antibody concentration of 0.2mg/ml - lot GR126616-1) and the loading control mouse anti-GAPDH antibody (ab8245; 1:10000) overnight at 4°C. Antibody binding was detected using infrared (IR) labelled goat anti-rabbit (green; 1:10000) and IR-goat anti-mouse (red; 1:10000) for 1 hour at room temperature before imaging.

ab15323 staining rat liver (lower image) and spleen (upper image) sections by IHC-P. The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Blocking of the sample was done with 1%BSA for 10 minutes at 21°C, followed by staining with ab15323 at 1/100 in TBS/BSA/azide for 16h at 21°C. A biotinylated goat anti-rabbit polyclonal antibody at 1/200 was used as the secondary antibody.

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ab15323 staining iNOS in Mouse NIH3T3 cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton and blocked with 10% serum for 30 minutes at 24°C. Samples were incubated with primary antibody (1/100) for 16 hours at 4°C. An Alexa Fluor® 488-conjugated Donkey anti-rabbit polyclonal was used as the secondary antibody (1/500).

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ab15323 staining iNOS in Mouse whole blood cells by Flow Cytometry. The sample was incubated with the undiluted primary antibody for 30 minutes at 25°C. ab97050, FITC-conjugated Goat anti-rabbit IgG (undiluted) was used as the secondary antibody.

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All lanes : Anti-iNOS antibody (ab15323) at 5 µg/ml

Lane 1 : Raw 264.7 whole cell lysate + PMA
Lane 2 : Raw 264.7 whole cell lysate + PMA + LPS + Brefeldin A

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 131 kDa
Additional bands at: 140 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes

All lanes : Anti-iNOS antibody (ab15323) at 5 µg/ml

Lane 1 : Human recombinant iNOS protein
Lane 2 : Mouse recombinant iNOS protein

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 131 kDa


Exposure time: 1 minute

Membrane was blocked in 2% milk for 1 hour at RT. Primary anitbody was incubated at 4°C overnight.

ab15323 staining macrophages in mouse spleen sections by IHC-P. The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Blocking of the sample was done with 1%BSA for 10 minutes at 21°C, followed by staining with ab15323 at 1/50 in TBS/BSA/azide for 16h at 21°C. A biotinylated goat anti-rabbit polyclonal antibody at 1/200 was used as the secondary antibody.

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