Anti-iNOS antibody

• WB

Lab

Western blot - Anti-iNOS antibody (AB15323)

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.

All lanes:

Western blot - Anti-iNOS antibody (ab15323) at 1 µg/mL

Lane 1:

Raw264.7 at 20 µg

Lane 2:

Raw264.7 + LPS (1 ug per mL; 18 hours) at 20 µg

Secondary

All lanes:

IR- goat anti-rabbit (green) at 1/10000 dilution

Predicted band size: 131 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody (AB15323)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse lung labelling iNOS with ab15323 at a concentration of 1µg/ml.

The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB kit and anti-rabbit HQ and anti-HQ HRP detection.
Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.
ab15323 Anti-iNOS antibody 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody (AB15323)

IHC image of iNOS staining in a section of formalin-fixed paraffin-embedded normal rat lung performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15323, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-iNOS antibody (AB15323)

IHC image of iNOS staining in a section of formalin-fixed paraffin-embedded normal mouse lung performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15323, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-iNOS antibody (AB15323)

All lanes:

Western blot - Anti-iNOS antibody (ab15323) at 5 µg/mL

Lane 1:

Raw 264.7 whole cell lysate + PMA at 20 µg

Lane 2:

Raw 264.7 whole cell lysate + PMA + LPS + Brefeldin A at 20 µg

Predicted band size: 131 kDa

Observed band size: 140 kDa

true

Exposure time: 8min

• WB

Lab

Western blot - Anti-iNOS antibody (AB15323)

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

All lanes:

Western blot - Anti-iNOS antibody (ab15323) at 5 µg/mL

Lane 1:

Human recombinant iNOS protein at 0.1 µg

Lane 2:

Mouse recombinant iNOS protein at 0.1 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 1/50000 dilution

Predicted band size: 131 kDa,47 kDa,68 kDa

true

Exposure time: 1min

• WB

CiteAb

Western blot - Anti-iNOS antibody (AB15323)

Western Blotting using Anti-iNOS antibody, ab15323. Publication image from Zhang, L. Y. et al., 2020, Theranostics, 31903107. Legend direct from paper.

C3aR inhibition suppresses microglial activation and microglia redistribution to myelin in BCCAO rats. A Western blots and quantification for CD86, iNOS, and β-actin in the striatum of the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 3-6 in each group. B Representative images of microglia cells (Iba-1+ cells, red) contacting with myelin (MBP+, green) in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. Scare bar=50 µm. n= 3-4 in each group. C-D Quantification of the proportion of microglia cells adhered to myelin relative to the number of myelin fibers (C) and the number of microglia cells (D). The data are shown as the mean ± SD. ***, p< 0.001; **, p< 0.01; *, p< 0.05; NS, not significant; the BCCAO group vs. control group. C3aRA : C3aR antagonist.

false

• WB

CiteAb

Western blot - Anti-iNOS antibody (AB15323)

Western Blotting using Anti-iNOS antibody, ab15323. Publication image from Zhang, L. Y. et al., 2020, Theranostics, 31903107. Legend direct from paper.

Genetic deletion of C3ar1 attenuates microglia activation and reverses white matter injury in BCAS mice. A Representative images and quantification of C3aR (green) and Iba-1 (red) double-positive cells and microglia cells (Iba-1+ cells, red) in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. Scale bar=25 µm. B Western blots and quantification for CD86, iNOS, MBP and β-actin in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. C Representative images and quantification of damaged axon (SMI32+, red) relative to myelin (MBP+, green) and mature oligodendrocyte (APC+ cells, red) in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. Scale bar=50 µm. D Western blots of total- and phospho-STAT3 (pSTAT3) and β-actin in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. E and F Quantification of total STAT3/ β-actin (E) and phospho-STAT3/STAT3 (F) levels in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. The data are shown as the mean ± SD. n = 3-4 animals in each genotype group. ***, p< 0.001; **, p< 0.01; *, p< 0.05; NS, not significant.

false

• WB

CiteAb

Western blot - Anti-iNOS antibody (AB15323)

Western Blotting using Anti-iNOS antibody, ab15323. Publication image from Zhang, L. Y. et al., 2020, Theranostics, 31903107. Legend direct from paper.

Aberrant activated microglia increase in BCCAO rats. A Representative images and quantification for the number of Iba-1+ microglia cells (red) in the striatum of control and BCCAO rats at day 7, 14, and 28 after surgery. n = 3-4 animals in each group. Scale bar=50 µm. B Quantitative RT-PCR analysis of the expression of Cd86, Cd16, and Inos in the striatum of control and BCCAO rats at day 7, 14, and 28 after surgery. The values are normalized to those of the control group. n = 3-6 in each group. C Western blots and quantification for CD86, CD16, iNOS, and β-actin in the striatum of BCCAO and control rats at day 7, 14, and 28 after surgery. n = 3 in each group. D Representative images and quantification of reactive microglia (CD86+ cells, red) in the striatum of BCCAO and control rats. n = 3-4 animals in each group. Scale bar=50 µm. The data are shown as the mean ± SD. ***, p< 0.001; **, p< 0.01; *, p< 0.05; NS, not significant; the BCCAO group vs. control group.

false