Tissue samples were fixed in 4% paraformaldehyde (EMS) at room temperature for 45 minutes, followed by an overnight incubation in 30% weight/volume sucrose solution (Sigma-Aldrich). Samples were then embedded in optimal cutting temperature (Tissue-Tek) and frozen on dry ice. Staining was performed on 10-μm sections by first blocking with 5% donkey serum and 0.1% Tween-20 for 1 hour, followed by overnight incubation with primary antibody diluted in blocking buffer in a humidified chamber. Sections were washed three times in PBS containing 0.1% Tween-20. For immunofluorescence staining, slides were then incubated with DAPI (Life Technologies, 1:1,000) and Alexa fluorophore–conjugated antibodies (Jackson ImmunoResearch). For IHC, slides were first incubated with biotinylated secondary antibodies (Jackson ImmunoResearch) and developed using the ABC HRP and DAB kits per manufacturer protocols (Vectorlabs). Primary antibodies used were as follows: rat anti–Ki-67 (eBioscience, 14–5698–82), rabbit anti–c-Myc (Y69; Abcam, Ab32072), rabbit anti-CD3 (Invitrogen, PA1–29547), rabbit anti-F4/80 (Novus, NBP2–12506), rat antineutrophil (Abcam, NIMP_R14), anti-CD206 (R&D Systems, AF2535), and rabbit anti-Arg1 (Cell Signaling Technology, 93668).
Rabbit anti arg1
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-Arg1 is a polyclonal antibody that specifically recognizes the Arg1 (Arginase-1) protein. Arg1 is an enzyme involved in the urea cycle and arginine metabolism. This antibody can be used to detect and quantify Arg1 expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Sucrose solution, by Merck Group (1 mentions)
Biotinylated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rat anti ki67, by Thermo Fisher Scientific (1 mentions)
Abc hrp and dab kits, by Vector Laboratories (1 mentions)
Ab32072, by Thermo Fisher Scientific (1 mentions)
Af2535, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rabbit anti ng2, by Abcam (1 mentions)
Rabbit anti gdnf, by Abcam (1 mentions)
Rabbit anti mbp, by Abcam (1 mentions)
Rabbit anti inos, by GeneTex (1 mentions)
Rabbit anti bdnf, by ABclonal (1 mentions)
Rabbit anti tubulin, by ABclonal (1 mentions)
Goat anti iba1, by Abcam (1 mentions)
Rabbit anti β actin, by ABclonal (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rabbit anti inos, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Mouse anti iba 1, by Cell Signaling Technology (1 mentions)
6 protocols using rabbit anti arg1
1
Multi-immunostaining of Tissue Sections
2
Western Blot Analysis of Neural Markers
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The concentration of protein was determined by BCA method to prepare protein loading samples. The brain protein extract (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to the PVDF membrane. The membrane was blocked with 5% skim milk on a shaker for 1 h. After washing with TBST, the membranes were incubated with the primary antibodies rabbit anti-MBP (1:500, Abcam), goat anti-Iba-1 (1:500, Abcam), rabbit anti-Arg-1 (1:800, Cell Signaling Technology), rabbit anti-iNOS(1:500, Genetex), rabbit anti-BDNF (1:500, Abclonal), rabbit anti-GDNF(1:500, Abcam), rabbit anti-CNTF (1:1000, Abcam), rabbit anti-NG2 (1:1000, Abcam), rabbit anti-β-actin (1:1000, Abclonal) and rabbit anti-Tubulin (1:1000, Abclonal) overnight at 4 °C. After washing with TBST, the membranes were added with the HRP–conjugated goat anti-rabbit (1:3000, Abclonal) and rabbit anti-goat (1:1000, Boster) second antibodies for 2 h at RT. After washing with TBST, immunoblots were developed with an enhanced chemiluminescence systemand measured using Quantity Software (Bio-Rad, Hercules, CA, USA).
3
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).
4
Protein Expression Analysis by Western Blot
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ARG1 and ARG2 expression was detected in cell lysates by SDS-PAGE followed by western blotting. K562 cells were lysed with Pierce RIPA Buffer (Thermo Scientific) with the Protease Inhibitor Cocktail (Sigma, Tokyo, Japan, #P8340). Extracted proteins (80 µg/well) were run on 10% acrylamide gel and transferred onto a nitrocellulose membrane for antibody staining and detection. The following primary monoclonal antibodies were used: rabbit anti-ARG1 (1:1000, Cell Signaling Technology, Danvers, MA, USA, #93668), rabbit anti-ARG2 (1:1000, Cell Signaling, #55003) and mouse anti-β-tubulin (1:10,000, Millipore, Burlington, MA, USA, 05-661). Also, goat anti-rabbit IgG (1:10,000, Cell Signaling Technology, #7074P2) or horse anti-mouse IgG (1:10,000, Cell Signaling Technology, #7076P2) HRP-conjugated secondary antibodies were used. Detection of the chemiluminescence signal was performed using the Clarity Max Western ECL Substrate (BioRad, Hercules, CA, USA). Before β-tubulin detection, the membranes were stripped with a buffer containing 1.5% glycine, 0.1% DSS and 1% Tween 20 (pH 2.2).
5
Protein Extraction and Western Blotting of Extracellular Vesicles
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Total protein was extracted from RAW264.7 cells, purified Nor-sEV and Hypo-sEV using RIPA lysis buffer with protease and phosphatase inhibitors (Thermo Fisher Scientific, USA). The protein concentration was quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA). For Western blot analysis of cell lysates, 30 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (SDS‒PAGE; Invitrogen, USA) and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% BSA (Sigma, USA) for 1 h at room temperature, followed by overnight incubation at 4 °C with the primary antibodies diluted in blocking solution according to the manufacturer's instructions. The following primary antibodies were used: mouse anti-CD9/63, mouse anti-TSG101 (Santa Cruz Biotechnology, CA, USA), rabbit anti-iNOS (Abcam, Cambridge, UK), rabbit anti-Arg1 (Cell Signaling Technology, MA, USA), mouse anti-NF-κB p105 and anti-p50 (Zen BioScience, Chengdu, China), and mouse anti-β-actin (Sigma‒Aldrich, USA). The membranes were then incubated for 1 h at room temperature in species-related horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Cell Signaling Technology, USA). The immunoreactive proteins were detected using a chemiluminescence kit (Millipore, USA).
6
Comprehensive Immunoblot Analyses for Cellular Phenotyping
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Western blot was performed as previously described.30 (link) The antibodies and dilution ratios were as follows: Rabbit anti-Arg1 (Cell Signaling Technology [CST] Danvers, MA; cat. no. 93668), rabbit anti-iNOS (CST; cat. no. 13120), mouse anti-Stat3 (CST; cat. no. 9139), rabbit anti-phospho-Stat3 (Tyr705) (CST; cat. no. 9145), mouse anti-E-cadherin (CST; cat. no. 14472), rabbit anti-N-cadherin (CST; cat. no. 13116), rabbit anti-Vimentin (CST; cat. no. 5741), rabbit anti-Cebpβ (Abcam; cat. no. ab32358), rabbit anti-ERK1/2 (CST; cat. no. 9102), rabbit anti-p-ERK1/2 (Thr202/Tyr204) (CST; cat. no. 9101), rabbit anti-S100A8 (CST; cat. no. 47310), rabbit anti-S100A9 (CST; cat. no. 73425), rabbit anti-IRF8 (CST; cat. no. 5628), and mouse anti-β-actin (Abcam, Cambridge, MA; cat. no. ab6276). All antibodies were used at a 1:1000 dilution, with the exception of mouse anti-β-actin, which was used at 1:5000.
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