Anti-iNOS 抗体

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody (AB3523)

Immunohistochemistry analysis of human heart tissue stained for iNOS without (negative control) or using ab3523 at 1/200 dilution overnight at 4°C in a humidified chamber, followed biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor.

To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-iNOS antibody (AB3523)

Immunofluorescence analysis of A549 (human lung carcinoma cell line) whole cells labelling iNOS (Left panel : green) without (control) or using ab3523 at 1/20 dilution overnight at 4°C, followed DyLight-488 conjugated secondary antibody. Counter stain : F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Cells were grown on chamber slides and fixed with formaldehyde prior to staining.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-iNOS antibody (AB3523)

Immunofluorescence analysis of NIH/3T3 (mouse embryo fibroblast cell line) whole cells labelling iNOS (Left panel : green) without (control) or using ab3523 at 1/20 dilution overnight at 4°C, followed DyLight-488 conjugated secondary antibody. Counter stain : F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Cells were grown on chamber slides and fixed with formaldehyde prior to staining.

• WB

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Western blot - Anti-iNOS antibody (AB3523)

This was run using 4-12% Bis-Tris Protein Gel and a Nitrocellulose membrane. Observed band at 130kDa in LPS treated RAW 264.7 cells, and an uncharacterized band at ~60kDa.

All lanes:

Western blot - Anti-iNOS antibody (ab3523)

Lane 1:

Whole cell lysate of RAW 264.7 at 30 µg

Lane 2:

Whole cell lysate of RAW 264.7 treated with LPS at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP

false

• WB

Supplier Data

Western blot - Anti-iNOS antibody (AB3523)

Using 4-20% Tris-Glycine polyacrylamide gel and transferred to a nitrocellulose membrane, blocked with 5% Milk in TBST for at least 1 hour. The membrane was probed with ab3523 at 4°C overnight on a rocking platform, washed in TBST,  and probed with the secondary antibody for 1 hour.

All lanes:

Western blot - Anti-iNOS antibody (ab3523)

Lane 1:

RAW264 whole cell lysate untreated at 20 µg

Lane 2:

RAW264 whole cell lysate untreated stimulated with LPS at 1 µg/mL for 16 hours at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (Heavy Chain) Superclonal Secondary Antibody, HRP conjugate at 1/1000 dilution

false