Anti nos2

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Spain

Anti-NOS2 is a laboratory reagent used for the detection and quantification of the enzyme Nitric Oxide Synthase 2 (NOS2) in biological samples. NOS2 is an important enzyme involved in the production of nitric oxide, a signaling molecule with diverse physiological functions. The Anti-NOS2 reagent can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and regulation of NOS2 in cells, tissues, or biological fluids.

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18 protocols using anti nos2

Visualizing Microglial Cell Activation

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Microglial cells were visualized with immunofluorescence, using rabbit polyclonal anti-ionized calcium binding adaptor molecule 1 (anti-IBA1; 1:400, Wako, Richmond, VA), a specific marker for microglia and macrophages [39 (link)]. To evaluate microglial cell activation, NOS2 expression was visualized using a mouse monoclonal anti-NOS2 (1:100; Santa Cruz Biotechnology, Heidelberg, Germany). Expression of VEGF-R1 and VEGF-R2 was evaluated with coimmunostaining with IBA1.
Briefly, after permeabilization with 0.1% Triton X-100 in PBS for 30 min, specimens were rinsed and saturated for 30 min with 5% goat serum in PBS. They were incubated overnight at 4 °C with the primary antibodies. After washing, sections were incubated for 1 h at room temperature with a secondary Alexa Fluor 488 (green)-conjugated goat anti-rabbit monoclonal antibody and a secondary Alexa Fluor 594 (red)-conjugated goat anti-mouse monoclonal antibody, each at dilution 1:200 (Invitrogen). For each step, antibodies were diluted in PBS-1% goat serum. After the nuclei were stained with DAPI (1:5,000; Sigma-Aldrich), the sections were mounted with gel mount and were observed with a fluorescence laser scanning microscope (Olympus BX51, Rungis, France).

Couturier A., Bousquet E., Zhao M., Naud M.C., Klein C., Jonet L., Tadayoni R., de Kozak Y, & Behar-Cohen F. (2014). Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation. Molecular Vision, 20, 908-920.

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Western Blot Analysis of TLR4, MyD88, and NOS2

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Cells were harvested, washed thrice with cold PBS followed by lysis in REPA lysis buffer containing 1% protease inhibitor cocktail (Sigma) and 3% phosphatise inhibitor cocktail (Sigma). Total protein concentration in cell lysate was evaluated using Bradford reagent (Sigma) and lysates containing equal amounts of protein (50 μg/well) were resolved by SDS PAGE and electroblotted to nitrocellulose membrane. Membranes were blocked in 5% w/v skimmed milk in 1X TBST for 30 minutes and incubated with rabbit polyclonal anti-TLR4 (1:1000 v/v), anti-MyD88 (1:1000 v/v), and anti-NOS2 (1:1000 v/v, all from Santa Cruz Biotechnology). Rabbit polyclonal anti–β-actin (1:500 v/v, Cell Signalling Technology), was used as loading control. Membranes were washed with 1xTBST and incubated with Goat anti-rabbit secondary antibody HRP conjugate (1:2000 v/v, polyclonal, Roche) for 2 h and visualized with Enhanced Chemiluminisence kit (Roche) according to manufacturer’s protocol.

Gupta P.K., Chakraborty P., Kumar S., Singh P.K., Rajan M.G., Sainis K.B, & Kulkarni S. (2016). G1-4A, a Polysaccharide from Tinospora cordifolia Inhibits the Survival of Mycobacterium tuberculosis by Modulating Host Immune Responses in TLR4 Dependent Manner. PLoS ONE, 11(5), e0154725.

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Western Blot Analysis of Signaling Proteins

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Cytosolic extracts were prepared as previously described [29 (link)]. Protein content was estimated by the Bio-Rad protein assay. Protein extracts were subjected to SDS–PAGE (10–15% gels) and blotted onto polyvinylidene difluoride membranes, which were incubated with the following antibodies: total MAPKs and phosphorylated forms of p38 and ERK (Cell Signaling Technology); with anti-NOS-2, anti-IκBα, anti-IκBβ, anti-β-actin, anti-p65, and anti-PSF (Santa Cruz Biotechnology); or with anti-COX-2 (Cayman Chemical). The blots were then incubated with secondary goat anti-mouse or rabbit IgG antibodies (Cell Signaling) and developed with ECL according to the manufacturer’s instructions (GE Healthcare). β-Actin and PSF were used as loading controls.

Cuadrado I., Amesty Á., Cedrón J.C., Oberti J.C., Estévez-Braun A., Hortelano S, & de las Heras B. (2018). Semisynthesis and Inhibitory Effects of Solidagenone Derivatives on TLR-Mediated Inflammatory Responses. Molecules, 23(12), 3197.

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Protein Expression Analysis via Western Blot

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Cells were washed and then lysed in the ice Complete Tablet buffer (Roche) supplemented with 2 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich), and 1 : 100 mix Protease Inhibitor Cocktail (Sigma-Aldrich). From each lysate, 35 μg proteins was resolved into 8% and 15% SDS-PAGE gels, and polyvinylidene difluoride (PVDF) membranes (GE Healthcare) were incubated overnight at 4°C with a specific primary antibody: anti-SOD3 (1 : 250, Santa Cruz), anti-phospho-PKAα/β/γ (Thr198, 1 : 250, Santa Cruz), anti-NOS2 (1 : 250, Santa Cruz), and anti-cytochrome C (1 : 1000, Calbiochem). Protein expression was normalized and verified through β-actin detection (1 : 5000; Sigma-Aldrich) and expressed as a mean ± SD (% vs. control).

Molinari C., Morsanuto V., Ghirlanda S., Ruga S., Notte F., Gaetano L, & Uberti F. (2019). Role of Combined Lipoic Acid and Vitamin D3 on Astrocytes as a Way to Prevent Brain Ageing by Induced Oxidative Stress and Iron Accumulation. Oxidative Medicine and Cellular Longevity, 2019, 2843121.

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Western Blot Analysis of Spinal Cord and Disc Tissues

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Spinal cord and lumbar disc 5–6 tissues were homogenized, and Western blots were performed as already described [79 (link),80 (link),81 (link)]. The samples were stored at −80 °C for further analysis.
Specific primary antibodies against Aggrecan (sc-166951, Santa Cruz Biotechnology), NGF (MA5-32067, Thermo Fisher, Milan, Italy), trkA (JJ084-04, Thermo Fisher), WNT3a (sc-80457, Santa Cruz Biotechnology), anti-FZ8 (Bioworld Technology, St. Louis Park, MN, USA), anti–β-catenin (610153, BD Biosciences), anti-active β-catenin (05-665, Millipore, Milan, Italy), anti-NFkB (Santa Cruz Biotechnology, sc-8008), anti-NOS2 (Santa Cruz Biotechnology, sc-7271), or anti-COX-2 (Santa Cruz Biotechnology, sc-376861) were mixed in a 5% w/v nonfat dried milk solution and incubated with the membranes at 4 °C overnight. Afterwards, the blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibodies or peroxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature [82 (link)]. The membranes were also incubated with antibodies against β-actin or lamin A/C (Santa Cruz Biotechnology) to verify that equal amounts of protein were loaded [83 (link)]. Images of the blot signals were imported into an analysis software (v2003, Image Quant TL, Milan, Italy) [84 (link)].

Genovese T., Impellizzeri D., D’Amico R., Cordaro M., Peritore A.F., Crupi R., Gugliandolo E., Cuzzocrea S., Fusco R., Siracusa R, & Di Paola R. (2022). Resveratrol Inhibition of the WNT/β-Catenin Pathway following Discogenic Low Back Pain. International Journal of Molecular Sciences, 23(8), 4092.

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Protein Extraction and Western Blot Analysis

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Total protein extracts were obtained after washing the hearts with PBS and adding 300 ml of RIPA modified lysis buffer (50 mM NaCl, 50 mM Tris–HCl (pH 7.40), 1% Triton X-100, 1 mM EDTA, 1 mM PMSF; 2.5 g/l Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO, USA), 1 mM Na₃VO₄, 1 mM NaF), or washing the cultured cells and scraped off the dishes with 50 µl of the same buffer. Then, the tubes were kept on ice for 30 min with swirling, and the samples were centrifuged at 7,000 g at 4°C for 10 min. The supernatants were stored at −20°C. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, USA) and bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) as a standard (36 (link)). For Western blot analysis, total proteins were boiled in Laemmli sample buffer, and equal amounts of protein (40–50 µg) were separated by 10–12% SDS-PAGE. The gels were blotted onto a Hybond-P membrane (GE Healthcare, Madrid, Spain) and incubated with the following antibodies: anti-NOS2, anti-NOS3, anti-Arginase I (Arg-I), anti-CD31, anti-VEGF-A, and anti-α-actin (Santa Cruz Biotechnology, CA, USA). The blots were revealed by enhanced chemiluminescence in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer’s instructions. Band intensity was analyzed using the NIH-ImageJ software (37 (link)).

Penas F.N., Carta D., Dmytrenko G., Mirkin G.A., Modenutti C.P., Cevey Á.C., Rada M.J., Ferlin M.G., Sales M.E, & Goren N.B. (2017). Treatment with a New Peroxisome Proliferator-Activated Receptor Gamma Agonist, Pyridinecarboxylic Acid Derivative, Increases Angiogenesis and Reduces Inflammatory Mediators in the Heart of Trypanosoma cruzi-Infected Mice. Frontiers in Immunology, 8, 1738.

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Immune Protein Expression Analysis

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After treatment with recombinant human IFN-γ and TNF-α (Peprotech) for 24 h, the cells were lysed in RIPA buffer (Thermo Fisher Scientific) with protease inhibitor. Protein concentrations were quantified using BCA assay kit (Thermo Fisher Scientific). Equal amounts of protein were separated by 10% SDS-polyacrylamide gel and analyzed with the following human antibodies: anti-IDO (Merk millipore), anti-TGF-β1 (Abcam), anti-NOS2 (Santa cruz Biotechnology, Dallas, TX, USA), anti-TSG-6 (R&D systems, Minneapolis, MN, USA), and anti-GAPDH (GeneTex, Irvine, CA, USA). The proteins were detected using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific) and luminescent image analysis LAS-3000 system (Fujifilm, Tokyo, Japan).

Kang J.Y., Oh M.K., Joo H., Park H.S., Chae D.H., Kim J., Lee H.R., Oh I.H, & Yu K.R. (2020). Xeno-Free Condition Enhances Therapeutic Functions of Human Wharton’s Jelly-Derived Mesenchymal Stem Cells against Experimental Colitis by Upregulated Indoleamine 2,3-Dioxygenase Activity. Journal of Clinical Medicine, 9(9), 2913.

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Western Blot Analysis of Inflammatory Proteins

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Proteins extracts were boiled in Laemmli sample buffer, and equal amounts of protein (50–100 mg) were separated by 8–12% SDS–PAGE. The gels were blotted onto a Hybond-P membrane (GE Health-care, Madrid, Spain) and incubated with the following antibodies: anti-NOS2, anti-IκB-α, anti-p65 (Santa Cruz Biotechnology, CA, USA), and anti-α-actin (Sigma–Aldrich Co). The blots were revealed by enhanced chemiluminescence (ECL) in an Image Quant 300 cabinet (GE Healthcare Biosciences, USA) following the manufacturer instructions. Band intensity was analysed using the Image J software.

Cevey Á.C., Mirkin G.A., Penas F.N, & Goren N.B. (2015). Low-dose benznidazole treatment results in parasite clearance and attenuates heart inflammatory reaction in an experimental model of infection with a highly virulent Trypanosoma cruzi strain. International Journal for Parasitology: Drugs and Drug Resistance, 6(1), 12-22.

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Western Blot Analysis of Inflammatory Markers

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Total air pouch membranes and RAW 264.7 cell extracts were homogenized and proteins were collected. Proteins were resolved on a 10% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. After blocking in 5% nonfat dry milk in Tris buffer-saline-Tween 20 (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 0.5% Tween 20), blots were incubated at room temperature with polyclonal anti-PGRN(1:1000 dilution, Santa Cruz Biotechnology), anti-COX-2(1:1000 dilution, Santa Cruz Biotechnology), anti-NOS-2(1:1000 dilution, Santa Cruz Biotechnology), anti-p65(1:000 dilution, cell signaling) anti-GAPDH (1:000 dilution, Santa Cruz Biotechnology)or anti-β-tubulin (1:000 dilution, Santa Cruz Biotechnology) for 1 h. After washing, the secondary antibody (horseradish peroxidaseconjugated anti-rabbit immunoglobulin; 1:3000 dilution) was added and incubated at room temperature for 1 hour, and bound antibody was visualized using an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL, USA).

Zhao Y.P., Wei J.L., Tian Q.Y., Liu A.T., Yi Y.S., Einhorn T.A, & Liu C.J. (2016). Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling. Scientific Reports, 6, 20909.

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Mycolactone Modulates Microglia Immune Responses

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The impact of mycolactone on TLR4 and IFNγR surface expression was measured in primary microglia exposed to increasing doses of mycolactone for 16 h. Microglia were detached with Accutase solution (Sigma) and blocked 20 min with FcR blocking reagent mouse (Mitenyi Biotec, 130-092-575) before being incubated 30 min with either a PE anti-mouse TLR4 (CD284)/MD2 complex antibody (117605, Biolegend) or a PE anti-mouse CD119 antibody (12-1191-82, ThermoFisher) or with the corresponding isotype in PBS 2% SVF at 4°C. SCs exposed to mycolactone for 16 h were detached with Accutase before being labeled with the PE anti-mouse TLR4 (CD284)/MD2 complex antibody. NOS-2 expression was monitored in microglia exposed to increasing doses of mycolactone, 30 min prior to LPS/ IFN-γ stimulation during 16 h (respectively 1 μg/ml and 20 ng/ml). Microglia were fixed (BD Lyse/ Fix buffer, BD Biosciences) and permeabilized (BD Perm/ Wash, BD Biosciences), then incubated for 30 min with anti-NOS2 (Santa Cruz Biotechnology, M-19), then for 20 min with the anti-goat DyLight 649 (Rockland, 605-443-002). Intracellular NOS2 staining was analyzed with the BD Accuri C6 flow cytometer (BD).

Isaac C., Mauborgne A., Grimaldi A., Ade K., Pohl M., Limatola C., Boucher Y., Demangel C, & Guenin-Macé L. (2017). Mycolactone displays anti-inflammatory effects on the nervous system. PLoS Neglected Tropical Diseases, 11(11), e0006058.

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