Rabbit anti inos

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-iNOS is a primary antibody that specifically binds to inducible nitric oxide synthase (iNOS), an enzyme involved in the production of nitric oxide in various cell types. This antibody can be used to detect and quantify the expression of iNOS in biological samples.

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18 protocols using rabbit anti inos

Investigating Inflammatory Pathways

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The antibodies used were: rabbit anti-TRPV4 (#ab39260, Abcam, Cambridge, Britain); rabbit anti-iNOS (#13120, Cell Signaling Technology, Danvers, MA, USA); mouse anti-F4/80 (#sc-377009, Santa Cruz, Santa Cruz, CA, USA); rabbit anti-COX2 (#ab15191, Abcam, Cambridge, Britain); rabbit anti-GAPDH (#8884, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-NLRP3 (#13158, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-CASPASE-1 (#22915-1-AP, Proteintech, Wuhan, China); and rabbit anti-ASC (#10500-1-AP, Proteintech, Wuhan, China).

Sun H., Sun Z., Xu X., Lv Z., Li J., Wu R., Fei Y., Tan G., Liu Z., Liu Y, & Shi D. (2022). Blocking TRPV4 Ameliorates Osteoarthritis by Inhibiting M1 Macrophage Polarization via the ROS/NLRP3 Signaling Pathway. Antioxidants, 11(12), 2315.

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Antibody Panel for Immune Response Analysis

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Antibodies used for Western blotting are as follows: rabbit anti-IL1α, rabbit anti-IL1β (Abcam ab9722), rabbit anti-IL6 (Novus Biologicals NB600-1131), rabbit anti-iNOS (Cell Signaling Technology CST 13120), rabbit anti-TNFα (Abcam ab9739) and mouse anti-VINCULIN (Sigma V9131). F4/80 antibody used for immunofluorescence and immunohistochemistry was purchased from eBiosciences (rat anti-mouse F4/80, clone BM8, ref 14-4801-82). CD16/CD32 antibody was purchased from BD Biosciences (rat anti-mouse-CD16/CD32, clone 2.4G2) and used at a concentration of 25 μg/ml. Monoclonal antibodies used for flow cytometry were all purchased from Biolegend unless indicated otherwise: anti-CD45 (Brilliant Violet 421-anti-mouse-CD45, clone 30-F11), anti-F4/80 (PE-anti-mouse-F4/80, clone BM8 or APC-anti-mouse-F4/80, clone BM8), anti-MHCII (FITC-anti-mouse-I-A/I-E, clone M5/114.15.2, BD Biosciences), anti-CD80 (PE/Cy7-anti-mouse-CD80, clone 16-10A1), anti-CD86 (PerCP-anti-mouse-CD86, clone GL-1), anti-IL1α (PE-anti-mouse-IL1α, clone ALF-161).

Cullis J., Siolas D., Avanzi A., Barui S., Maitra A, & Bar-Sagi D. (2017). Macropinocytosis of nab-paclitaxel drives macrophage activation in pancreatic cancer. Cancer immunology research, 5(3), 182-190.

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Immunohistochemical Analysis of Spinal Cord Injury

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Frozen coronal sections (7 μm) were collected near the collagenase injection site and washed three times in PBS at room temperature. Antigen retrieval was performed by placing slides in a pressure cooker containing citrate buffer (pH 6.0) for 4 min. Tissues were permeabilized by 1% triton-X100 in PBS for 30 min, washed in PBS, and blocked in 1% BSA in PBS for 1 hr. Slides were incubated overnight at 4°C in 1% BSA containing the following primary antibodies: mouse anti-3-NT (1:500, Sigma), rabbit anti-iNOS (1:300, Cell Signaling), rabbit anti-GFAP (1:1000, Abcam), mouse anti-Iba1 (1:1000, Wako), rabbit anti-CD31 (1:300, EMD Millipore), and mouse anti-NeuN (1:1000, Sigma). The slides were washed in PBS, and then incubated for 1 hr at room temperature in 1% BSA containing Alexa Fluor®594-conjugated goat anti-rabbit and DyLight^™488-conjugated goat anti-mouse (1:500, Jackson ImmunoResearch) secondary antibodies. After a final PBS wash, slides were mounted with SlowFade® Diamond Antifade Mountant with DAPI (ThermoFisher) and a coverslip. Images were captured using fluorescent microscopy. Mean fluorescence intensity and cell count per field were quantified by a person who was blinded to the experimental groups, using NIH image J software.

Bahader G.A., Nash K.M., Almarghalani D.A., Alhadidi Q., McInerney M.F, & Shah Z.A. (2021). Type-I Diabetes Aggravates Post-Hemorrhagic Stroke Cognitive Impairment by augmenting Oxidative Stress and Neuroinflammation in Mice. Neurochemistry international, 149, 105151.

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Western Blot Analysis of Mitochondrial Proteins

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Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokoy, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk in Tris-buffered saline (TBS), and probed using various antibodies. The Western blots were visualized using ECL (Bio-rad) and exposed to Amersham™ Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: mouse anti-Mfn2 (1:500, Abcam); mouse anti-HO-1 (1:500, Abcam); rabbit anti-PGC1α (1:1000, Abcam); rabbit anti-iNOS (1:1000; Cell Signaling); rabbit anti-Nrf2 (1:1000, Abcam); rabbit anti-pink1 (1:500, Novus Biologicals, CO, USA); mouse anti-parkin (1:1000, Cell Signaling); mouse anti-β-actin (1:1000, Santa Cruz); horseradish peroxidase-conjugated anti-rabbit or -mouse antibodies (1:2500, Abcam).

Hong J.Y., Kim H., Jeon W.J., Baek S, & Ha I.H. (2020). Antioxidative Effects of Thymus quinquecostatus CELAK through Mitochondrial Biogenesis Improvement in RAW 264.7 Macrophages. Antioxidants, 9(6), 548.

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Astragalus Polysaccharides Modulate Macrophage Phenotype

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Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA); Astragalus polysaccharides (APS, UV ≥ 98%) were bought from Yuan-ye Biotechnology (Shanghai, China). RPMI-1640 medium, penicillin-streptomycin solution, 0.05% trypsin-digestion solution, and fetal bovine serum were obtained from Gibco (Grand Island, NY, USA). Phosphate buffer (PBS) was purchased from HyClone (South Logan, UT, USA). Rabbit anti-GAPDH, rabbit anti-iNOS, rabbit anti-Arg-1, mouse anti-IBA-1, anti-rabbit IgG HRP-linked antibody, and anti-mouse IgG HRP-linked antibody were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). Goat anti-mouse IgG H + L antibody was obtained from Biotech (Beijing, China).

Liu X., Ma J., Ding G., Gong Q., Wang Y., Yu H, & Cheng X. (2021). Microglia Polarization from M1 toward M2 Phenotype Is Promoted by Astragalus Polysaccharides Mediated through Inhibition of miR-155 in Experimental Autoimmune Encephalomyelitis. Oxidative Medicine and Cellular Longevity, 2021, 5753452.

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Antibody Characterization for Cellular Analysis

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The following antibodies were used: mouse anti-F4/80 (Santa Cruz), rabbit anti-iNOS (Cell Signaling Technology), mouse anti-CD80 (Invitrogen Antibodies), rabbit anti-CD206 (Abcam), rabbit anti-SHP2 (Cell Signaling Technology), rabbit anti-COL2 (Abcam), rabbit anti-COL10 (Abcam), rabbit anti-MMP3 (Proteintech), rabbit anti-COX2 (Cell Signaling Technology), rabbit anti-GAPDH (Cell Signaling Technology), rabbit anti-p-P65 (Cell Signaling Technology), rabbit anti-P65 (Cell Signaling Technology), rabbit anti-β-actin (Cell Signaling Technology), rabbit anti-histone H3 (Cell Signaling Technology), rabbit anti-p-AKT (Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), and rabbit anti-p-IKKα/β (Cell Signaling Technology).

Sun Z., Liu Q., Lv Z., Li J., Xu X., Sun H., Wang M., Sun K., Shi T., Liu Z., Tan G., Yan W., Wu R., Yang Y.X., Ikegawa S., Jiang Q., Sun Y, & Shi D. (2022). Targeting macrophagic SHP2 for ameliorating osteoarthritis via TLR signaling. Acta Pharmaceutica Sinica. B, 12(7), 3073-3084.

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Western Blot Quantification Protocol

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Ten percent SDS-PAGE was used to separate protein samples (about 80 μg protein), which were then transferred to PVDF membranes (Millipore, Billerica, MA, USA). PVDF membranes were then blocked by 5% BSA for 1 h at 37°C. PVDF membranes were cut off in accordance with the molecular weight, and incubated with different primary antibodies (rabbit anti-GAPDH, 1:1000 dilution; mouse anti-RAGE, 1:800; rabbit anti-p-NF-κB P65, 1:800; mouse anti-GFAP, 1:1000; rabbit anti-Iba-1, 1:800; mouse anti-p-JNK, 1:800; rabbit anti-PARP, 1:1000 from Santa Cruz Biotechnology; rabbit anti-COX2, 1:1000; rabbit anti-iNOS, 1:1000 from Cell Signaling Technology) at 4°C overnight. On the second day, PVDF membranes were incubated with HRP-conjugated secondary antibody (anti-mouse and anti-rabbit, 1:2000) for 2 h at 37°C. Protein bands were detected by an ECL western blotting kit using a ChemiDoc-It™ imaging system (UVP, Upland, CA, USA). GAPDH was selected as a control. The gray value was analyzed by Gel-pro 32 (Media Cybernetics, Rockville, MD, USA).

Lian W., Jia H., Xu L., Zhou W., Kang D., Liu A, & Du G. (2017). Multi-Protection of DL0410 in Ameliorating Cognitive Defects in D-Galactose Induced Aging Mice. Frontiers in Aging Neuroscience, 9, 409.

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Immunohistochemical Analysis of AGEs, iNOS, Caspase-3, and NF-κB

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Immunohistochemical staining was performed according to previously reported protocols [33 (link)]. The antibodies used in this study were mouse anti-AGEs (6D12, Cosmo bio, Tokyo, Japan), rabbit anti-iNOS (Cell Signaling, Denver, MA, USA), rabbit anti-cleaved caspase-3 (Cell Signaling) and mouse anti-NF-κB (MAB3026, Chemicon International, Temecula, CA, USA). To detect AGEs, iNOS and NF-κB, the slides were labeled with a LSAB kit (DAKO, Carpinteria, CA, USA) and visualized with a DAB substrate kit (DAKO). For the detection of cleaved caspase-3, the sections were incubated with tetramethylrhodamine-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and detected by fluorescence microscopy (BX51, Olympus, Tokyo, Japan). For the morphometric analyses, the immunoreactive intensity per unit area (mm²) was measured using an ImageJ software (NIH, Bethesda, MD, USA).

Kim J., Jo K., Lee I.S., Kim C.S, & Kim J.S. (2016). The Extract of Aster Koraiensis Prevents Retinal Pericyte Apoptosis in Diabetic Rats and Its Active Compound, Chlorogenic Acid Inhibits AGE Formation and AGE/RAGE Interaction. Nutrients, 8(9), 585.

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Western Blot Analysis of Inflammatory Markers

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Proteins were extracted from the retinas of C57BL/6 mice at specific time points and adjusted for protein content (40 µg). Protein samples were then mixed with 5× SDS sample buffer (Beyotime), separated by SDS polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA). After being blocked in 5% nonfat dried milk for 2 hours at room temperature, the membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies used in this study included rabbit anti-IκBα, rabbit anti-p-IκBα, rabbit anti-p65, rabbit anti-p-p65, rabbit anti-iNOS, mouse anti-β-actin (Cell Signaling Technology), rabbit anti-interleukin-1β (IL-1β), rabbit anti-Toll-like receptor (TLR)2 (Abcam), goat anti-CD206, rabbit anti-transforming growth factor-β (TGF-β), rabbit anti-TLR4 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Arg-1 (GeneTex), goat anti IL-1RA, goat anti-monocyte chemoattractant protein1 (MCP-1; R&D Systems, Minneapolis, MN, USA). Membranes were washed and incubated for 2 hours with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology). We used enhanced chemiluminescence (ECL) western blotting detection solutions (EMD Millipore) to display the immunoreactive bands.

Sui A., Chen X., Demetriades A.M., Shen J., Cai Y., Yao Y., Yao Y., Zhu Y., Shen X, & Xie B. (2020). Inhibiting NF-κB Signaling Activation Reduces Retinal Neovascularization by Promoting a Polarization Shift in Macrophages. Investigative Ophthalmology & Visual Science, 61(6), 4.

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Quantification of Nitric Oxide and iNOS

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The determination NO₂ in cells supernatants was carried out using the quantitative Nitric Oxide Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the instructions by the manufacturer_. The determination of iNOS was made as follows: Cells were lysed on ice in lysis buffer (1% NP-40, 50 mN Tris-HCl, pH 7.6, 150 mM NaCl and 2 mM EDTA) and clarified by centrifugation at 5000× g for 5 min in a microcentrifuge at 4 °C. In addition, LPS infected macrophages were used as the positive control for this experiment. Proteins were separated by SDS-PAGE and transferred to polyvinylidine fluoride (PVDF) membranes (Biorad, Hercules, CA, USA). Antibodies used were rabbit anti-iNOS (Cell Signaling, Danvers, MA, USA) and goat anti-actin (Abcam, Cambridge, UK). Chemiluminescence was detected using the enhanced chemiluminescent (ECL) reagents (GE Healthcare, Chicago, IL, USA). To determine iNOS expression, densitometry was performed using Adobe Photoshop.

Kadir N.A., Acosta A., Sarmiento M.E, & Norazmi M.N. (2020). Immunomodulatory Effects of Recombinant Mycobacterium smegmatis Expressing Antigen-85B Epitopes in Infected J774A.1 Murine Macrophages. Pathogens, 9(12), 1000.

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