IHC was performed as previously described [64 (link)]. Briefly, protein expression in the TNBC samples was evaluated using a 1:200 dilution of mouse-monoclonal (clone 4E5) iNOS Antibody (MA5-17139 Invitrogen). The intensity of the staining received a score of 0–3 if the staining was negative, weak, moderate, or strong. The TMAs were scored by two pathologist and consensus reached.
Inos antibody
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The INOS antibody is a laboratory reagent used for the detection and quantification of inducible nitric oxide synthase (iNOS) in various biological samples. It is designed to specifically bind to the iNOS protein, enabling researchers to measure its expression and activity levels. The INOS antibody is a valuable tool for studies related to inflammation, immune response, and other physiological processes involving nitric oxide signaling.
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Lab products found in correlation
Trypsin edta, by Merck Group (2 mentions)
Phenol, by Merck Group (2 mentions)
Glycerol, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Carrageenan, by Merck Group (2 mentions)
Il 1β, by Santa Cruz Biotechnology (2 mentions)
Tnf α, by Santa Cruz Biotechnology (2 mentions)
Ammonium sulphate, by Merck Group (2 mentions)
Hematin, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit, by Abcam (1 mentions)
F4 80 antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Alexa fluor 594 goat anti rat, by Abcam (1 mentions)
Cox 1, by Merck Group (1 mentions)
Cox 2 enzymes, by Merck Group (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
5 lox, by Santa Cruz Biotechnology (1 mentions)
Diethyldithiocarbamate, by Merck Group (1 mentions)
7 protocols using inos antibody
1
Immunohistochemical Analysis of iNOS in TNBC
2
Immunofluorescent Analysis of Tumor Macrophages
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For the immunofluorescent study, the mice were sacrificed 12 h after macrophages injection, and the tumor tissues were dissected and fixed by 4% paraformaldehyde. To permeabilize the membranes, the frozen sections (10 μm) of tumor tissues were cut and treated with 0.3% Triton X-100 in PBS for 10 min. After washing in PBS, the tissue sections were incubated overnight at 4 °C with F4/80 antibody (1:100, Abcam, UK) or iNOS antibody (1:50, Invitrogen, USA) for 24 h, then treated with secondary antibodies Alexa Fluor® 488 goat anti-rabbit (Abcam, USA) or Alexa Fluor® 594 goat anti-rat (Abcam, USA) for 2 h. And 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, USA) at a concentration of 0.1 μg/mL in PBS was used to counterstain nuclei. F4/80, iNOS, and DAPI were represented as red, green, and blue colors, respectively. The fluorescent cell images were captured with a Slide scanner Axio Scan Z1 (ZEISS, Germany).
3
Optimizing Celecoxib-Mangiferin Synergy Assessment
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MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), Tris, Ethylene diamine tetra acetic acid (EDTA), Trypsin-EDTA, Di-ethyl dithio-carbamate (DDC), Tween-20, Hematin, Glycerol, Phenol, Ammonium sulphate, Carrageenan, COX-1 and COX-2 enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA). Celecoxib was a generous gift from Unichem Laboratories (Mumbai, India). The stock solutions of PT-Mangiferin (100 mg/ml) was prepared in dimethyl sulphoxide and further dilutions were made in PBS for treatment in vitro and in vivo. Polyclonal antibodies to, COX-2, IL-1β and TNF-α were purchased from Santa Cruz Biotechnology (California, U.S.A.). iNOS antibody was from Thermo scientific. All other chemicals and solvents were of analytical grade and purchased from authorized standard companies.
4
Isoorientin Extraction and Characterization
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MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), trypsin-EDTA, Tris, ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DDC), Tween-20, hematin, glycerol, phenol, and ammonium sulphate were purchased from Sigma Chemical Co. (St. Louis, MO). Celecoxib was a generous gift from Unichem Laboratories (Mumbai, India). Carrageenan was purchased from Sigma-Aldrich (St. Louis, USA). Polyclonal antibodies to COX-2, IL-6, 5-LOX, IL-1β, and TNF-α were purchased from Santa Cruz Biotechnology (California, USA). iNOS antibody was from Thermo Fisher Scientific Inc. All other chemicals and solvents were of analytical grade and purchased from authorized standard companies. Isoorientin was isolated from the methanolic extracts of tubers of P. tuberosa [13 (link)].
5
Paeonol and Methotrexate Combination Protocol
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The paeonol was obtained from Sigma-Aldrich (St. Louis, MO, USA), and the MTX was obtained from Minapharm Pharmaceuticals (Cairo, Egypt). Kits for determining serum ALT and AST were purchased from Biodiagnostic (Giza, Egypt). In addition, the PCNA antibody was procured from Novus Biologicals (Centennial, CO, USA), the iNOS antibody was from Thermo Fisher Scientific (Waltham, MA, USA), the TNF-α antibody was from ABclonal (Woburn, MA, USA), while the P-gp and Mrp-2 antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). The other chemicals were purchased from local commercial sources and were of analytical grade.
6
Macrophage Cholesterol Loading and iNOS Analysis
7
Isolation and Characterization of Activated Lung Macrophages
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Mice were sacrificed and lungs were aseptically removed. To prepare a single-cell suspension, lungs were minced and digested in 5% FBS/PBS solution containing 250 U/ml collagenase IV (Worthington Biochemical Corporation) and 20 U/ml DNase (Roche) for 60 min at 37°C. Lung tissues were then passed through a 70-µm cell strainer, and red blood cells were lysed with ammonium-chloride-potassium buffer.
For cell sorting, lung cell suspensions were blocked with anti–mouse CD16/32 followed by incubation with CD11b (BD) and CD80 (eBioscience) antibodies. CD11b+ mCherry+ CD80high cells (activated population) and CD11b+ mCherry+ CD80low cells (resting population) were sorted using a cell sorter (S3; Bio-Rad Laboratories) with a 100-µm nozzle. Cells were then surface stained with CD11b (BD), CD80 (eBioscience), CD40 (eBioscience), CD86 (eBioscience), and MHCII (eBioscience). For iNOS staining, surface-stained cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer set (eBioscience). Cells were then incubated for 30 min with an iNOS antibody (eBioscience). Data were acquired using a flow cytometer (LSR II; BD) and analyzed with FlowJo software (Tree Star).
For cell sorting, lung cell suspensions were blocked with anti–mouse CD16/32 followed by incubation with CD11b (BD) and CD80 (eBioscience) antibodies. CD11b+ mCherry+ CD80high cells (activated population) and CD11b+ mCherry+ CD80low cells (resting population) were sorted using a cell sorter (S3; Bio-Rad Laboratories) with a 100-µm nozzle. Cells were then surface stained with CD11b (BD), CD80 (eBioscience), CD40 (eBioscience), CD86 (eBioscience), and MHCII (eBioscience). For iNOS staining, surface-stained cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer set (eBioscience). Cells were then incubated for 30 min with an iNOS antibody (eBioscience). Data were acquired using a flow cytometer (LSR II; BD) and analyzed with FlowJo software (Tree Star).
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