Anti-iNOS antibody

• IHC-P

Supplier Data

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

Supplier Data

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

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• 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

• WB

CiteAb

Western blot - Anti-iNOS antibody (AB3523)

Western Blotting using Anti-iNOS antibody, ab3523. Publication image from Saini, V. et al., 2020, Nat Commun, 31992699. Legend direct from paper.

Interaction between •NO and H2S and pharmacological modulation of sulfide levels in mice.a Representative western blot and b densitometry analysis of iNOS in mouse lungs at 8 weeks post infection (n = 3 animals per group). c Nitrate and nitrite levels in the sera of infected (Inf) and uninfected mice and d the culture medium of infected mouse macrophages exposed to 25 µM GYY4137 for 36 h. Data in (c) and (d) are pooled from two independent experiments (n = 7−8 animals per group). e OCR profile and f average OCR of Mtb cells exposed to •NO (DETA/NO; 250 µM), •NO followed by GYY4137 (10 µM), or untreated (UT) in XF media (n = 6−8). Media control contained no cells. g ATP levels (shown as RLU normalized to CFU) in Mtb cells treated (•NO, GYY4137, or •NO and GYY4137) or untreated (UT) for 2 or 8 h. h CFUs after 8 and 24 h of exposure. T = 0 indicates CFU at the start of the experiment. Data in (g) and (h) are representative of at least two independent experiments. i CFU recovered from infected Cbs+/+ peritoneal macrophages receiving one of the following : aminooxyacetic acid (AOAA), AOAA followed by GYY4137, GYY4137 alone, or untreated (UT) (n = 4). jMtb CFU recovered from mouse peritoneal macrophages. k Bacillary burden in the lungs of mice that received : AOAA, AOAA followed by GYY4137, or GYY4137 alone. AOAA (13 mg/kg, 6 d/wk for 6 weeks) and GYY4137 (25 mg/kg 3 d/wk for 6 weeks) were administered intraperitoneally. Treatment began 2 weeks post-Mtb infection and continued for 6 wks. Data represent two independent experiments (n = 5−6 animals per group). l Total sulfide levels in serum of mice in (k) using the methylene blue method (n = 6−8). Data represent mean ± SEM except in (e, f and l) (mean ± SD). Comparisons are against WT mice or untreated (UT) controls. Data were analyzed by unpaired parametric t test (b, j), one-way ANOVA with Tukey’s multiple comparisons test (c, d, f, k and l) or two-way ANOVA with Sidak’s multiple comparisons test (g−i). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

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