Anti-iNOS antibody [RM1017]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] (AB283655)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling iNOS with ab283655 at 1/2000 (0.3 μg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used. Positive staining on the hepatocytes in human liver. The section was incubated with ab283655 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] (AB283655)

Immunohistochemical analysis of formalin fixed paraffin embedded human lung labelling iNOS with ab283655 at a concentration of 0.01µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab283655 anti-iNOS antibody [RM1017] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] (AB283655)

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling iNOS with ab283655 at 1/2000 (0.3 μg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used. Positive staining on the human lung carcinoma. The section was incubated with ab283655 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-iNOS antibody [RM1017] (AB283655)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Caco-2 (Human colorectal adenocarcinoma epithelial cell) cells labelling iNOS with ab283655 at 1/600 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] (AB283655)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling iNOS with ab283655 at a concentration of 0.01µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab283655 anti-iNOS antibody [RM1017] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] (AB283655)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling iNOS with ab283655 at 1/2000 (0.3 μg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used. Positive staining on the human lung. The section was incubated with ab283655 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IP

Lab

Immunoprecipitation - Anti-iNOS antibody [RM1017] (AB283655)

iNOS was immunoprecipitated from DLD-1 (human colorectal adenocarcinoma epithelial cell) treated with 100U/mL IFN gamma (human), 50U/mL IL-1 beta (human) and 10ng/mL TNF alpha for 24h whole cell lysate with ab283655 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283655 at 1/2000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : DLD-1 (human colorectal adenocarcinoma epithelial cell) treated with 100U/mL IFN gamma(human), 50U/mL IL-1 beta(human) and 10ng/mL TNF alpha for 24h whole cell lysate
Lane 2 : ab320005 IP in DLD-1 (human colorectal adenocarcinoma epithelial cell) treated with 100U/mL IFN gamma(human), 50U/mL IL-1 beta(human) and 10ng/mL TNF alpha for 24h whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283655 in DLD-1 treated with 100U/mL IFN gamma (human), 50U/mL IL-1 beta (human) and 10ng/mL TNF alpha for 24h whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-iNOS antibody [RM1017] (ab283655) at 1/2000 dilution

All lanes:

DLD-1 (human colorectal adenocarcinoma epithelial cell) treated with 100U/mL IFN gamma(human), 50U/mL IL-1 beta(human) and 10ng/mL TNF alpha for 24h whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 131 kDa

Observed band size: 131 kDa

false

Exposure time: 32s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-iNOS antibody [RM1017] (AB283655)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml LPS for 6h (Red) / Untreated control (Green) cells labelling iNOS with ab283655 at 1/600 dilution (0.1ug) (Red) and Green (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-iNOS antibody [RM1017] (AB283655)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling iNOS with ab283655 at 1/50 (12.0 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 μg/ml) dilution (Green). Confocal image showing cytoplasmic staining in RAW 264.7 cell line after treatment with lipopolysaccharide ( 1ug/ml ) for 6 h. is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5μg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 μg/ml) dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-iNOS antibody [RM1017] (AB283655)

iNOS was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1μg/ml LPS for 6h whole cell lysate 10μg with ab283655 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283655 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml LPS for 6h whole cell lysate 10μg

Lane 2 : ab283655 IP in RAW 264.7 treated with 1μg/ml LPS for 6h whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283655 in RAW 264.7 treated with 1μg/ml LPS for 6h whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 8 seconds

Lower bands could be proteolytic cleavage as described in literature. (PMID : 11786228)

All lanes:

Immunoprecipitation - Anti-iNOS antibody [RM1017] (ab283655)

Predicted band size: 131 kDa

false

• WB

Lab

Western blot - Anti-iNOS antibody [RM1017] (AB283655)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-iNOS antibody [RM1017] (ab283655) at 1/2000 dilution

Lane 1:

DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

DLD-1 treated with 100U/mL IFN gamma(human), 50U/mL IL-1 beta(human) and 10ng/mL TNF alpha for 24h whole cell lysate at 20 µg

Lane 3:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 5:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 6h whole cell lysate at 20 µg

Lane 6:

NR8383 (rat alveolar macrophage) whole cell lysate at 20 µg

Lane 7:

NR8383 treated with 1ng/ml lipopolysaccharide (LPS) for 24h whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 131 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-iNOS antibody [RM1017] (AB283655)

Blocking buffer and concentration : 5% NFDM/TBST iNOS is not normally expressed in the brain, but can be induced in the brain after inflammatory, infectious or other damages (PMID : 11138926, PMID : 16156895, PMID : 10322315).

Lanes 1 - 3:

Western blot - Anti-iNOS antibody [RM1017] (ab283655) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-iNOS antibody [RM1017] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/inos-antibody-rm1017-bsa-and-azide-free-ab283668'>ab283668</a>)

Lane 1:

Mouse hippocampus tissue lysate at 20 µg

Lane 2:

Mouse colon tissue lysate at 20 µg

Lane 3:

Mouse colon cancer tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 131 kDa

false

• I-ELISA

Supplier Data

Indirect ELISA - Anti-iNOS antibody [RM1017] (AB283655)

Indirect ELISA using ab283655 at varying antibody concentrations and antigen concentration at 1000 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.