Immunofluorescence staining was performed as previously described with minor modification [28 (link)] [PMID: 33,472,663]. The lung tissue or THP-1 cell sections were blocked with 1% bovine serum albumin (BSA) (Sangon Biotech, Shanghai, China) for 15 min and incubated with anti-CD68 (diluted 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-CD86 (diluted 1:100; ABclonal Technology, Wuhan, China)), anti-iNOS (diluted 1:100; Affinity Biosciences, Changzhou, China), anti-NLRP3 (diluted 1:100; ABclonal Technology, Wuhan, China), anti-ASC (diluted 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-p65 (diluted 1:400; CST, Danvers, MA, USA) overnight. Subsequently, the slices were incubated with corresponding secondary antibodies (Cy3: goat-anti rabbit, diluted 1:200, Invitrogen, Carlsbad, CA, USA; FITC: goat-anti-mouse, diluted 1:200, Abcam, Cambridge, UK), followed by counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI) (Aladdin, Shanghai, China). Fluorescence microscope (Olympus, Tokyo, Japan) was used to capture stained images.
Anti inos
Manufactured by Affinity Biosciences
Sourced in China, United States
Anti-iNOS is a laboratory reagent used to detect and quantify the expression of inducible nitric oxide synthase (iNOS) in biological samples. iNOS is an enzyme responsible for the production of nitric oxide, which plays a crucial role in various physiological and pathological processes. Anti-iNOS is a specific antibody that can be used in techniques such as Western blotting, immunohistochemistry, and ELISA to measure iNOS levels in cell cultures, tissue extracts, or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti asc, by Santa Cruz Biotechnology (1 mentions)
Anti cd68, by Santa Cruz Biotechnology (1 mentions)
Anti nlrp3, by ABclonal (1 mentions)
Anti cd86, by ABclonal (1 mentions)
Goat anti rabbit cy3, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse fitc, by Abcam (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Anti cd206, by Affinity Biosciences (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Affinity Biosciences (1 mentions)
Anti cd68, by Affinity Biosciences (1 mentions)
Ripa lysis buffer, by Fudebio (1 mentions)
Anti il10, by ABclonal (1 mentions)
Anti cd86, by Affinity Biosciences (1 mentions)
Enhanced bca protein assay kit, by Proteintech (1 mentions)
Anti ho1, by Huabio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti nrf2, by Proteintech (1 mentions)
3 protocols using anti inos
1
Immunofluorescence Staining of Lung and THP-1 Cells
2
Western Blot Analysis of Macrophage Markers
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We used the RIPA Lysis Buffer (Fudebio, Hangzhou, China) containing phosphatase/protease inhibitors (Fudebio, Hangzhou, China) to extract total protein from the cultured cells following the manufacturer’s method. SDS‐polyacrylamide gel electrophoresis (10%; SDS‐PAGE) was performed to separate the protein samples (20 µg), which were then transferred to Millipore polyvinylidene difluoride (PVDF) membranes. These PVDF membranes were kept in overnight incubation at 4°C with anti‐CD206, anti‐ODC, anti‐IL‐33, anti‐iNOS, anti‐CD68, anti‐ARG1 and anti‐GADPH primary antibodies (Affinity, USA), followed by incubation at room temperature for 60 minutes with horseradish peroxidase (HRP)–conjugated secondary antibodies (Affinity, USA) that were diluted to 1:5000 in 5% skim milk. After washing with TBST, the membranes were treated for 1 minutes with SuperSignal™ West Dura Extended Duration Substrate (Fudebio, Hangzhou, China) and were visualized through chemiluminescence.
3
Protein Expression Analysis in Mouse Spinal Cord
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Following the experimental procedures, mice were deeply anesthetized and sacrificed. Spinal dorsal horn (L3-L5) tissues were promptly dissected, and RIPA lysis buffer (Beyotime, China) along with protease inhibitors got supplemented. Total protein was extracted using the lysis buffer. After centrifugation at 12,000 g and 4°C for 30 min, subsequently harvesting supernatant. We detected protein concentration utilizing an enhanced BCA protein assay kit (Proteintech, Wuhan, China). Equivalent amounts of protein got loaded onto 10‑15% percent SDS-PAGE gel for electrophoresis gel for different protein detections, subsequently transferred onto PVDF membranes (Millipore, MA). Submerging PVDF membranes with 5% milk for 2h at room temperature, they subsequently got exposed to primary antibodies at 4°C for a whole night. After having the membranes washed, they were sent for incubation with appropriate secondary antibodies, and subjected to detection by ECL Western blotting detection system. The following primary antibodies were used: anti-Nrf2 (1:2000, Proteintech), anti-HO-1 (1:2000, Hua-bio), anti-iNOS (1:1000, Affinity), anti-CD86 (1:1000, Affinity), anti-IL-10 (1: 2000, ABclonal), anti-Arg-1 (1:1000, Affinity) and anti-β-actin (1:2000, Proteintech).
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