All infected and uninfected tissues were dissociated by collagenase digestion as described previously 40 (link). Cell suspensions were stained and analyzed by flow cytometry. The following antibodies were used for cell surface staining: anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD3 (145-2C11), anti-Ly6C (HK1.4), anti-MHCII (M5/114.15.2), anti-CD19 (ebio1D3), anti-NK1.1 (PK136), anti-siglecH (ebio440c), anti-B220 (RA3-6B2), anti-CD4 (GK.5), anti-CD8 (53-6.7) and anti-CD69 (H1.2F3) were purchased from eBioscience, anti-Ly6G (1A8) was obtained from BD Pharmingen, anti-Ly6-B2 antibody (7/4) from AbD Serotec, and CCR2 (475301) from R&D Systems. For intracellular staining: anti-iNOS antibody was purchased from Millipore, and anti-TNF-α (MP6-XT22) was purchased from BD Pharmingen. Cell suspensions from collagenase-digested tissue were stained for cell surface markers and permeabilized using the BD Cytofix/Cytoperm Kit (BD Pharmingen). For detection of intracellular TNF-α, cells were also treated ex vivo with GolgiPlug (BD Pharmingen) for 6 h at 37°C in complete RPMI medium prior to cell surface staining and stained according to the manufacturers instructions. Acquisitions were performed with a BD LSRII, and data were analyzed with the FlowJo software.
Anti inos antibody
Manufactured by Merck Group
Sourced in United States
The Anti-iNOS antibody is a laboratory tool used to detect the presence and quantity of the inducible nitric oxide synthase (iNOS) protein in biological samples. iNOS is an enzyme involved in the production of nitric oxide, which plays a role in various physiological and pathological processes. The antibody allows researchers to measure iNOS levels and monitor its expression in cells or tissues, providing a valuable tool for studying the involvement of nitric oxide signaling in different biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd69 h1.2f3, by Thermo Fisher Scientific (2 mentions)
Anti cd8 53 6, by Thermo Fisher Scientific (2 mentions)
Anti ly6g 1a8, by BD (2 mentions)
Ccr2 475301, by R&D Systems (2 mentions)
Anti tnf α mp6 xt22, by BD (2 mentions)
Anti ly6 b2 antibody 7 4, by Bio-Rad (2 mentions)
Lsr 2, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Trypsin ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer 2x, by Bio-Rad (1 mentions)
Lipopolysaccharide lps from salmonella enterica serotype typhimurium, by Merck Group (1 mentions)
Enhanced chemiluminescence ecl kit, by Biosesang (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
8 protocols using anti inos antibody
1
Flow Cytometry Analysis of Immune Cell Populations
2
Western Blot Analysis of Nitric Oxide Synthases
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Samples were homogenized in lysis buffer on ice, then centrifuged at 12,000 rpm for 10 min (4°C). Supernatants were collected and protein concentration was determined using a BCA assay. Proteins (5 μg) were separated by 10% SDS-PAGE under reducing conditions and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Bio-sciences, Freiburg, Germany). Membranes were blocked in a TBST (Tris-buffer saline containing 0.05% Tween-20) solution containing 5% nonfat milk and then probed (4°C; overnight) with an anti-nNOS antibody (1:500, Epitomics, Burlingame, CA, USA), an anti-iNOS antibody (1:500, Millipore Corp, billerica, MA, USA), or an anti-GAPDH antibody (1:1000, Sigma Chemical Co, St. Louis, MO, USA), diluted in TBST containing 5% nonfat milk. Following washes in TBST buffer, horseradish peroxidase-conjugated secondary antibody (1:2000, ZSGB-BIO, ORIGENE, Beijing, China) was used to detect primary antibodies (2 h at room temperature). ECL plus reagent was used to develop films and the intensity of bands was determined by Gel-Pol analyzer.
3
Flow Cytometry Analysis of Immune Cell Populations
4
Investigating LPS-Induced Inflammatory Responses
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The lipopolysaccharide (LPS) from Salmonella enterica serotype typhimurium and the Griess reagent (G4410) were purchased from Sigma-Aldrich Chemical Co. (USA). The anti-iNOS antibody was purchased from Millipore (USA), and anti-β-actin antibody was purchased from Santa Cruz Biotechnology (USA).
We procured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ammonium pyrrolidinedithiocarbamate (APDTC), protease inhibitor cocktail from Merck (Germany). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/ streptomycin, trypsin-ethylenediaminetetraacetic acid, BCA protein assay kit were purchased from Thermo Fisher Scientific (USA). Dimethyl sulfoxide (DMSO), radioimmunoprecipitation (RIPA) buffer, and enhanced chemiluminescence (ECL) kit were purchased from Biosesang (Korea) and Laemmli sample buffer (2X) was purchased from Bio-Rad (USA).
We procured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ammonium pyrrolidinedithiocarbamate (APDTC), protease inhibitor cocktail from Merck (Germany). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/ streptomycin, trypsin-ethylenediaminetetraacetic acid, BCA protein assay kit were purchased from Thermo Fisher Scientific (USA). Dimethyl sulfoxide (DMSO), radioimmunoprecipitation (RIPA) buffer, and enhanced chemiluminescence (ECL) kit were purchased from Biosesang (Korea) and Laemmli sample buffer (2X) was purchased from Bio-Rad (USA).
5
Western Blot Analysis of Neuroinflammatory Markers
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For western blotting analysis, tissue extracts were prepared by homogenizing mouse hippocampus tissue in T-PER Tissue Protein Extraction Reagent (Thermo Scientific Inc., Waltham, MA, USA) containing protease inhibitor, α-complete (Roche Applied Science, Penzberg, Germany). Fifteen micrograms of each tissue extract sample were adjusted to give a final solution of 60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5% β-mercaptoethanol. The solution was heated at 100ºC for 5 min, electrophoresed through a 10% SDS-polyacrylamide gel, and transferred to polyvinylidine difluoride membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Plexin-A1, Neuropilin-1, COX-2, iNOS, IL-1β, TNF-α and β-actin were detected by their respective antibodies using an enhanced chemiluminescence western blot detection system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Anti-Plexin-A1 antibody (Abcam), anti-Neuropilin-1 antibody (Abcam), anti-COX-2 antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-iNOS antibody (Merck Millipore, Darmstadt, Germany), anti-IL-1β antibody (Santa Cruz Biotechnology), anti-TNF-α antibody (Santa Cruz Biotechnology), and anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA) were used.
6
Immunoblotting Analysis of iNOS Protein
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Treated-RAW 264.7 cells were collected and lysed with RIPA buffer containing protease inhibitors. The protein samples were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane. The target proteins were probed with specific antibodies, an anti-iNOS antibody at a dilution of 1:2000 (Merck, Rahway, NJ, USA), and an anti- β-actin antibody at a dilution of 1:10,000 (Sigma, St. Louis, MO, USA). Protein detection was carried out using a chemiluminescence reagent.
7
Quantitative Measurement of iNOS
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The iNOS contents were determined using the method described previously by Marshall et al. [29 (link)] and modified by Saia et al. [18 (link)]. The macrophage homogenates were diluted in phosphate-buffered saline (PBS 0.1 M, pH 7.2) and incubated overnight at 4°C in Nunc Maxisorb plates (Life Technologies, Paisley, UK). The wells were washed with PBS containing 0.05% Tween-20 (PBS-T) and blocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, the polyclonal rabbit anti-iNOS antibody (1:1000; Sigma) was added for 1 h at room temperature. After washing, the biotinylated anti-rabbit IgG secondary antibody (1:200; Vector Laboratories) was added to each well, incubated with avidin-HRP (1:5000; Sigma) for 30 min, and the colour developed by addition of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; BD Biosciences, Franklin Lakes, NJ, USA). The reaction was stopped with 2 N H2SO4 and the optical density (OD) reading at 450 nm was taken. The sample levels of iNOS were expressed as OD normalized to the respective total protein concentrations.
8
Inhibition of LPS-induced iNOS and COX-2 in RAW 264.7 cells
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RAW 264.7 cells were seeded at a density of 5 × 105 cells/mL in a 6-well plate. After overnight incubation, they were pretreated with different concentrations of ETMI for 1 h before the addition of LPS (0.5 μg/mL). After incubation for 24 h, the cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer. Western blotting was performed as previously described 19 (link). Anti-iNOS antibody was purchased from Sigma-Aldrich (Cat# N7782), and anti-COX-2 and anti-β-actin antibodies were obtained from Cell Signaling (Cat# 4842 and Cat# 3700, respectively). Immunoblot signals were compared with those for β-actin and the relative protein expression was determined.
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