Anti-iNOS antibody [RM1017] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB283668)

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] (<a href='/en-us/products/primary-antibodies/inos-antibody-rm1017-ab283655'>ab283655</a>) 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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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 ( 1μg/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 ug/ml dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1μg/ml LPS for 6h (Red) / Untreated control (Green) cells labelling iNOS with ab283655 at 1/600 dilution (0.1μg) (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.

• IP

Supplier Data

Immunoprecipitation - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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 1μg/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] (<a href='/en-us/products/primary-antibodies/inos-antibody-rm1017-ab283655'>ab283655</a>)

Predicted band size: 131 kDa

false

• WB

Lab

Western blot - Anti-iNOS antibody [RM1017] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/inos-antibody-rm1017-ab283655'>ab283655</a>) 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] - BSA and Azide free (AB283668)

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). This data was developed using ab283655, the same antibody clone in a different buffer formulation.

Lanes 1 - 3:

Western blot - Anti-iNOS antibody [RM1017] (<a href='/en-us/products/primary-antibodies/inos-antibody-rm1017-ab283655'>ab283655</a>) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-iNOS antibody [RM1017] - BSA and Azide free (ab283668)

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] - BSA and Azide free (AB283668)

This data was developed using ab283655, the same antibody clone in a different buffer formulation.

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.