To identify the polarization states of macrophages (M1 co-expressing F4/80 + iNOS and M2 co-expressing F4/80 + ARG-1), double staining immunofluorescence was performed. After blocking with 3% BSA (Thermo Fisher Scientific, Inc.) at room temperature for 30 min, THP-1 cells were incubated with anti-F4/80 (cat. no. sc-377009; dilution, 1:100; Santa Cruz Biotechnology, Inc.), anti-iNOS (cat. no. sc-7271; dilution, 1:100; Santa Cruz Biotechnology, Inc.) and anti-ARG-1 (cat. no. #93668; dilution, 1:50; Cell Signaling Technology, Inc.) antibodies. Next, THP-1 cells were washed with PBS and incubated with goat anti-mouse IgG H&L (Alexa Fluor® 488) secondary antibody (cat. no. ab150113; dilution, 1:200; Abcam) at room temperature for 1 h in the dark. Next, the cells were washed with PBS and incubated with DAPI at room temperature for 10 min in the dark. Finally, THP-1 cells were observed and photographed under a fluorescence microscope (Olympus Corporation).
Anti arg1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Arg1 is an antibody product developed by Santa Cruz Biotechnology. It is designed to recognize and bind to the Arg1 (Arginase 1) protein, which is involved in the regulation of cellular metabolism. The core function of this product is to provide a tool for the detection and study of Arg1 in various research and experimental applications.
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Lab products found in correlation
Anti inos, by Santa Cruz Biotechnology (4 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti stat6, by Cell Signaling Technology (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 or 568, by Thermo Fisher Scientific (1 mentions)
A1rmp multiphoton confocal microscope, by Nikon (1 mentions)
Anti inos, by R&D Systems (1 mentions)
Opal 4 color manual ihc kit, by PerkinElmer (1 mentions)
Anti cd68, by Abcam (1 mentions)
Anti inos, by Thermo Fisher Scientific (1 mentions)
Iba 1, by Abcam (1 mentions)
Alex fluor 488 labeled secondary antibody, by Abcam (1 mentions)
Eos digital camera, by Canon (1 mentions)
Alex fluor 647 labeled secondary antibody, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated igg, by MultiSciences Biotech (1 mentions)
12 protocols using anti arg1
1
Macrophage Polarization Immunostaining Protocol
2
Immunofluorescence Analysis of BV2 Microglial Cells
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BV2 microglial cells were grown on the coverslips fixed with 4% PFA for 15 min at RT and washed three times with PBS. Cells were blocked in blocking buffer (5% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS) for 1 h at RT. Cells were then incubated with the following primary antibodies overnight at 4 °C: anti-CD11b (monoclonal mouse; 1:250, Abcam), anti-Arg1 (polyclonal rabbit; 1:200; Santa Cruz) and then secondary antibodies conjugated with Alexa Fluor® 488 or 568, (1:500, Life Technologies, California, United States) were used. Finally, coverslips were washed three times in PBS, stained with 4′-6-diamidino-2-phenlyindole (DAPI, Sigma-Aldrich) 5 µg/mL in PBS, and mounted in fluorescent mounting medium (SHANDON, ThermoFisher Scientific Inc., Waltham, MA, USA). Fluorescence images were captured with Zeiss AxioImager M2 482 epifluorescence microscope equipped with a 483 AxioCam HRm camera. Images were acquired with the AxioVision4 software. For fluorescent intensity measuring, 8-bit grayscale images were taken at 10× magnification. Mean fluorescence values were measured in 6-OHDA-treated BV2 cells with or without rhCDNF administration, and after background subtraction, the values for 6-OHDA-treated BV2 cells were normalized to those of BV2 cells with PBS treatment. In vitro experiments were performed and analyzed 6–7 times.
3
Multicolor IHC Analysis of Bone Marrow
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Formalin‐fixed paraffin‐embedded bone marrows were analyzed using the Opal 4‐Color Manual IHC Kit (catalog no. NEL810001KT, Perkin Elmer) per the manufacturer's instructions. A Nikon A1RMP+ multiphoton confocal microscope was used to capture the fluorescent images. The primary antibodies were anti‐CD68 (catalog no. ab222914, Abcam), anti‐iNOS (catalog no. MAB9502, R&D Systems), and anti‐ARG1 (catalog no. sc166920, Santa Cruz).
4
Immunohistochemical Analysis of Neuroinflammation
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The sections were dewaxed, rehydrated, and then immersed into citrate buffer for antigen repair. After that, the sections were blocked with 2% bovine serum albumin and then incubated overnight at 4 °C with the following primary antibodies: anti-iNOS (1:200; Invitrogen, USA), anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:100, Abcam, USA), anti-Arg-1 (1:200, Santa Cruz Biotechnology, USA), followed by incubation with the secondary antibodies Alex Fluor® 488-labeled secondary antibody (green; 1:1000; Abcam), Alex Fluor® 647-labeled secondary antibody (red; 1:1000; Abcam) and Hoechst (blue; 1:1000) for 1 h at room temperature. Slides were observed by a fluorescent microscope (Olympus IX-71) equipped with a Canon EOS digital camera. The iNOS, Arg-1, and Iba-1 positive cells were separately counted using Image J software.
5
Western Blot Analysis of Immune Markers
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Protein extracts were subjected to SDS-PAGE (8–12% gels) and blotted onto PVDF membranes. After blocking with 5% fat-free milk, the membranes were incubated with the following antibodies: anti-AKT, anti-p-AKT, anti-Arg-1, anti-GAPDH, anti-β-actin (all purchased from Santa Cruz Biotechnology), anti-STAT6, anti-p-STAT6, anti-STAT3, anti-p-STAT3(705) (all purchased from Cell Signaling Technology), anti-Ym1, anti-IL10 (all purchased from Abcam) at 4 °C overnight. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated IgG (MULTI Sciences) and visualized with enhanced chemiluminescence (ECL, PerkinElmer) detection reagents (Thermo scientific, USA). β-actin or GAPDH was used as a loading control. IL-10 concentration in plasma, tissues, and cell culture supernatants were determined by ELISA kit (DAKEWE). The tissue ELISA measurements were normalized to the protein content of the homogenates.
6
qPCR and Western Blot Analysis of Immune Markers
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The qPCR was performed as previously described [63 (link)]. The total volume of 20 μl contained 2 μl of cDNA, 10 μl of 2×SYBR Premix Ex Taq, 7 μl ddH2O and 10 μmol/l of the primer pairs. The sequences of the used primers were listed in the supplement Table 1 and qPCR was performed using Roche LightCycler480 II. The western blotting was performed as previously described [64 ]. Primary antibodies included anti-IL-4 antibody (Santa Cruz, sc-53084), anti-IL-4Rα antibody (Santa Cruz, sc-28361), anti-Nrf2 (Abcam, ab137550), anti-p-Nrf2 antibody (Abcam, ab76026), anti-Nos2 (Santa Cruz, sc-7271), anti-Arg1 (Santa Cruz, sc-18355), and anti-β-actin (Cell Signaling Technology, 4970S).
7
Lung Tissue Protein Profiling
8
Immunohistochemical Staining of Brain Tissue
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Nitric acid, sodium azide, paraformaldehyde, urea, citric acid and sodium citrate were obtained from Fluka and Sigma (Sigma-Aldrich, St Louis, MO, USA). Biotinylated tomato lectin (TL), normal goat serum, the Mouse on Mouse (MOM) reagent kit, and the streptavidin/biotin blocking kit were obtained from Vector Laboratories (Burlingame, CA, USA). The following primary antibodies were used: anti-ionized calcium binding adaptor molecule 1 (Iba1, Wako, Osaka, Japan); anti-iNOS (Santa Cruz Biotechnology, Dallas, TX, USA); anti-Arg1 (Santa Cruz Biotechnology); anti-ubiquitin (Dako, Carpinteria, CA, USA); and anti-dephosphorylated neurofilament heavy and medium chain (SMI32, Covance, Princeton, NJ, USA) (Table 1 ). AlexaFluor-conjugated and biotin-conjugated secondary antibodies, AlexaFluor-conjugated streptavidin, and Nuclear Yellow dye were obtained from Molecular Probes (Life Technologies, Carlsbad, CA, USA) (Table 1 ).
9
Comprehensive Western Blotting Protocol
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Western blotting was routinely performed as previously reported [31 (link)]. Briefly, brain tissues were quickly dissected on ice and placed in RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail and phosphatase inhibitor (Beyotime, Shanghai, China). Denatured proteins in SDS-loading buffer were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, United States). After blocking, the membranes were incubated overnight at 4 °C with primary antibodies involving anti-occludin (Invitrogen), anti-ZO-1 (Invitrogen), anti-TNF-α (Santa Cruz), anti-iNOS (Proteintech), anti-Arg1 (Santa Cruz), anti-TRPM4 (Sigma-Aldrich) and anti-β-actin (Proteintech). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (Proteintech), and bands were detected by enhanced chemiluminescence advance Western blotting detection reagents (FDbio, Hangzhou, China). The intensities of the protein bands were quantified and normalized to the level of β-actin by using ImageJ software.
10
Western Blot Analysis of Inflammatory Markers
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