Coronal sections were incubated with 10% normal donkey serum for 30 minutes at room temperature in PBS containing 0.1% Triton X‐100 followed by incubation with appropriate primary antibodies overnight at 4°C in the same buffer. The anti‐Iba‐1(1:500, Wako), anti‐Arg‐1 (1:500, Abcam), anti‐CD206(1:300, Abcam), and anti‐iNOS (1:300, BD Biosciences) primary antibodies were used. After primary antibody incubation, sections were washed four times at room temperature, followed by incubation with appropriate fluorescent‐labeled secondary antibodies (1:1000) for 1 hour at room temperature. DAPI was incubated for counterstaining of the nucleus. Sections were then washed with PBS and mounted with water‐based mounting medium containing antifading agents. All the confocal images were captured on a laser scanning confocal microscope (Olympus Fluoview FV3000, Olympus). The numbers of target immunopositive cells were quantified by a blinded investigator using NIH Image J (1.52a). Three randomly selected microscopic fields in the cortex on each section were analyzed for each brain by a blinded investigator. The immunopositive cell was presented as the mean percentage of cells per field (CD206 and iNOS measured at 40X magnification, Arg‐1 measured at 80X magnification).
Anti arg 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Arg-1 is an antibody that detects the arginase-1 protein. Arginase-1 is an enzyme involved in the urea cycle and is expressed in hepatocytes. The antibody can be used to identify and quantify arginase-1 in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti inos, by Abcam (9 mentions)
Anti cd206, by Abcam (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti il 1β, by Abcam (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti iba1, by Fujifilm (1 mentions)
Fluoview fv3000, by Olympus (1 mentions)
Anti inos, by BD (1 mentions)
Elivisiontmplus kit, by Maixin Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti vegfa, by Proteintech (1 mentions)
Anti atf4, by Abcam (1 mentions)
Goat anti rabbit igg hrp antibody, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Trypan blue solution, by Solarbio (1 mentions)
16 protocols using anti arg 1
1
Quantification of Microglial Phenotypes
2
Immunohistochemical Analysis of ARG1
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EliVisionTMplus kit (Maixin, China) was used for immunohistochemistry assay in accordance with the manufacturer's instructions [11 (link)]. The anti-ARG1 was purchased from Abcam (USA). Determination of the ARG1 immunostaining score was made by the sum of staining intensity and positive stained cells rate. The staining intensity was graded as follows: no staining (0); weak staining (1); moderate staining (2); and strong staining (3). The positive stained cells rate was graded as follows: 0 ~ 5% (0); 5% ~ 25% (1); 26% ~ 50% (2); 51% ~ 75% (3); and >75% (4). The final score was the sum of the two sets of scores, and the score lower than 2 was regarded as negative staining, ≤ 3 as ARG1 low expression, and >3 as ARG1 high expression.
3
Western Blot Analysis of Protein Expression
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Proteins were obtained by RIPA lysis buffer (Beyotime, China) and the concentrations were assessed by BCA assay (Beyotime, China). After being separated by 10% SDS-PAGE, proteins were transformed onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% skim milk for two hours and then incubated with the following primary antibodies overnight at 4 °C: anti-Atf4 (Abcam, 1:1000), anti-Vegfa (Proteintech, 1:1000), anti-iNOS (Abcam, 1:1000), anti-Arg1(Abcam, 1:1000), anti-β-actin (Proteintech, 1:1000). After three washes, the membranes were incubated with secondary antibodies for one hour at room temperature, and the blots were captured by Tanon 5200 luminous imaging system. The results were quantified using the Image J software.
4
Morroniside Alleviates LPS-Induced Inflammation
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Forty-eight BALB/c mice (half male and half female; body mass 22–28 g) were purchased from the Experimental Animal Center of Lanzhou University (No. SCXK (Gan) 2018-0002). The mice were housed at a temperature of (25 ± 1)°C, relative humidity of 65% ± 10%, and a 12 hr light–dark cycle. All studies were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and in compliance with the regulations of the Gansu Provincial Animal Management Committee. All necessary efforts were made to minimize animal suffering and reduce the number of animals used in the experiments.
Lipopolysaccharide and morroniside were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Anti-iNOS, anti-COX2, anti-Arg-1, anti-CD206, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3, and goat anti-rabbit IgG-HRP antibodies were purchased from Abcam (Cambridge, UK). TNF-α, IL-6, and IL-1β ELISA kits were purchased from Shanghai ZCIBIO Technology Co., Ltd. PBS and trypan-blue solution were purchased from Beijing Solarbio Technology Co., Ltd. A bicinchoninic acid assay (BCA)-100 Protein Quantitative Analysis Kit was purchased from Shanghai Biocolor Biotechnology Co., Ltd. RIPA and BCA protein assay kits were purchased from Beyotime, Beijing. ECL was purchased from Affinity, Shanghai. AG490 was purchased from Shanghai Zerun Bio.
Lipopolysaccharide and morroniside were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Anti-iNOS, anti-COX2, anti-Arg-1, anti-CD206, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3, and goat anti-rabbit IgG-HRP antibodies were purchased from Abcam (Cambridge, UK). TNF-α, IL-6, and IL-1β ELISA kits were purchased from Shanghai ZCIBIO Technology Co., Ltd. PBS and trypan-blue solution were purchased from Beijing Solarbio Technology Co., Ltd. A bicinchoninic acid assay (BCA)-100 Protein Quantitative Analysis Kit was purchased from Shanghai Biocolor Biotechnology Co., Ltd. RIPA and BCA protein assay kits were purchased from Beyotime, Beijing. ECL was purchased from Affinity, Shanghai. AG490 was purchased from Shanghai Zerun Bio.
5
Western Blotting Analysis of YAP, CTGF, Arg1, and iNOS
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Western blotting was performed in a standard procedure. Tissue or cell lysates were prepared in RIPA buffer containing complete protease inhibitor cocktail and inhibitor for phosphatase. Protein concentration was determined using a BCA protein assay kit (Abcam, Shanghai, China). Proteins were then separated by a SDS-PAGE system. Membranes were incubated with primary antibodies including anti-YAP (Cell Signaling, MA, USA)), anti-CTGF (Abcam, Shanghai, China), anti-Arg1 (Abcam, Shanghai, China) and anti-iNOS (Abcam, Shanghai, China) at 4°C overnight and were probed with HRP-labeled secondary antibody. The membranes were visualized using an enhanced chemiluminescence system (Kodak, Rochester, USA). β-actin was used as a loading control.
6
Western Blot Analysis of Signaling Proteins
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The treated cells were mechanically homogenized and centrifuged at 3000 g for 30 min at 4°C to collect supernatant. The protein extracts were denatured and were heated for 10 min at 98°C. The samples (40 μg/sample) were then loaded on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) for electrophoresis and transferred to a PVDF membrane. The membrane was incubated with blocking solution for 1 hr and probed with the following primary antibodies: anti-α2-AR (Antibodies.com), anti-iNOS, anti-Arg1, anti-Ym1 (Abcam), anti-PPAR (this antibody and below were all from Cell Signaling Technology), anti-Akt, anti-p-Akt (Ser473), anti-p-PTEN (Ser380), anti-PTEN, anti-p-GSK-3β (Ser9), anti-p-PDK (Ser241), anti-STAT6, and anti-IRF-4 in 1:1000 dilution overnight at 4°C, followed by HRP-conjugated secondary antibody for 1 hr. The loading control was probed with anti-GADPH antibody (1:10,000, Sigma-Aldrich). The membrane was washed with TBST and visualized with an enhanced chemiluminescence (ECL) system (Santa Cruz, UK). The protein bands were captured with an image processor (GeneSnap; SYNGENE®, Cambridge, UK).
7
Immunofluorescence Microscopy for Macrophage Polarization
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Immunofluorescence microscopy was performed as previously described using an anti-α-SMA primary antibody (1:1000, Abcam) and Ce6-conjugated secondary antibody.15 (link) After staining, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 20 min. The cells were imaged using an A1R-AI confocal microscope (Nikon, Tokyo, Japan) using laser excitation of 543 nm for the detection of Ce6 fluorescence. Immunofluorescence staining of tissue sections was also conducted to evaluate the effect of Exo-MSCsIL−1β on macrophage polarization. After antigen retrieval, tissue sections were incubated with an anti-Arg-1 (1:1000, Abcam) antibody at 4°C overnight. Goat anti-rat IgG H&L (Alexa Fluor 647, Abcam) was applied for 30 min incubation at 37°C. DAPI was used to counterstain the nucleus. Finally, confocal microscopic pictures were obtained.
8
Curcumin-Collagen Hydrogel for Osteoarthritis
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Gelatin type B, tragacanth gum, Curcumin, Glacial acetic acid, Sodium sulfate, 37% formaldehyde, Chondroitin sulfate, EDC, NHS were purchased from Soleberg Reagent. Collagen (Type II, Chicken, Protein 60%, Yuanye), Fuchsin Complete Adjuvant (FCA), Fuchsin Incomplete Adjuvant (FICA) was purchased from Genye Reagent Company. Nitrendipine was obtained from Aladdin Chemical Reagent Co. CCK-8 kit, ROS kit, malondialdehyde (MDA) content assay kit, total glutathione (GSH-Px) content assay kit, superoxide dismutase (SOD) content assay kit and H&E kit were purchased from Beyotime Anti-HIF-1α, Anti-iNOS, anti-Arg-1, anti-IL-1β, anti-IL-10 were purchased from Abcam. RAW264.7 cells were obtained from Chinese Academy of Sciences.
9
Western Blot Analysis of Exosomal and Macrophage Proteins
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The treated exosomes or macrophages were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), and total protein was extracted after centrifugation. After the quantitative analysis, the protein in the appropriate loading buffer was denaturated by boiling at 100 °C for 10 min. Next, the proteins were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. After sealing, anti-TSG101 (Abcam, Cambridge, UK), anti-CD81 (Abcam, Cambridge, UK), anti-Arg-1 (Abcam, Cambridge, UK), and anti-iNOS (Abcam, Cambridge, UK) were added to the membranes that were kept at 4 °C overnight. The membrane was then washed and exposed to secondary antibodies for 1 h. After ECL coloring, each band was exposed and developed.
10
Arg-1 and Hpx Protein Analysis in Spinal Cord Injury
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Spinal cord tissue (1 mm above and below the lesion site, ~ 2 mm length) from the lesion site (T8–T9) was homogenized in RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Tissue lysates were subjected to western blotting using an anti-Arg-1 (Abcam, Cambridge, MA) or anti-Hpx (Abcam, Cambridge, MA) antibody. Protein bands were analyzed and quantified using densitometry and an Image-Pro Plus analysis system (Media Cybernetics, Silver Spring, MD) by normalizing their levels to the levels of GAPDH bands.
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