Islets were harvested, fixed in 4% paraformaldehyde, and immersed in 30% sucrose solution to be embedded in a frozen block. Inflammatory markers and dedifferentiation markers were detected in cell sections by immunofluorescence. Anti-FoxO1 (Santa Cruz, Dallas, Texas, USA), anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA), anti-Oct4 (Abcam, Cambridge, UK), anti-NGN3 (Abcam), anti-A11 oligomer (Invitrogen), anti-Pdx1 (Abcam), anti-IL-1β (Abcam), and anti-insulin (Abcam) were used as primary antibodies. M1/M2 macrophage markers were identified using the antibodies of M2 macrophage differentiation markers (anti-arginase-1; Cell Signaling) and M1 macrophage differentiation markers (CD80; Abcam). Sections were incubated with primary antibody diluted in blocking buffer overnight at 4°C. Secondary antibodies (Alexa Fluor® 488-conjugated goat anti-rabbit, Alexa Fluor® 488-conjugated goat anti-mouse, and Alexa Fluor® 594-conjugated goat anti-rabbit; Abcam) were added and incubated for 1 h at room temperature. Counterstaining was performed using DAPI (1 : 10,000). Images were obtained from each section using a fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).
Anti arginase 1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Arginase-1 is a research-use-only antibody product provided by Cell Signaling Technology. It is designed to detect arginase-1, an enzyme involved in the urea cycle.
Automatically generated - may contain errors
Lab products found in correlation
Anti inos, by Cell Signaling Technology (4 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Anti β actin, by Yeasen (2 mentions)
Anti foxo1, by Santa Cruz Biotechnology (1 mentions)
Anti oct4, by Abcam (1 mentions)
Anti pdx1, by Abcam (1 mentions)
Anti insulin, by Abcam (1 mentions)
Anti ngn3, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Universal hood 2 imager, by Bio-Rad (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Macrophage isolation kit peritoneum, by Miltenyi Biotec (1 mentions)
Anti bdnf, by Cell Signaling Technology (1 mentions)
9 protocols using anti arginase 1
1
Islet Inflammatory and Dedifferentiation Analysis
2
Macrophage Protein Expression Analysis
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Peritoneal cells were isolated immediately after the infected and control mice were sacrificed under sterile conditions 9 months post-infection. Macrophages were separated using a Macrophage Isolation Kit (Peritoneum) (Miltenyi Biotec, Bergisch Gladbach, Germany) and the purity (F4/80+) exceeded 90%, as determined by flow cytometry. Macrophages and non-macrophage cells were respectively lysed for 30 min on ice in a RIPA solution containing protease inhibitors. Lysates were then separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). After blocking of non-specific binding sites, the respective blots were incubated with different primary antibodies [anti-Arginase-1, anti-Arginase-2, anti-iNOS and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA)] and the respective HRP-conjugated secondary antibodies. The results were visualized using the ECL detection system (Merck Millipore) and recorded with a Universal Hood II Imager (Bio-Rad, California, USA). Band intensities were evaluated using image J (NIH, Bethesda, MD, USA).
3
Protein Expression Analysis of Brain Tissue
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The brains were homogenized on ice with a Ultrasonic crusher (HD3000, Wiggens, Beijing, China) in ice-cold lysis buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, RIPA) supplemented with protease inhibitors PMSF. Lysates were centrifuged at 10,000 × g for 20 min at 4 °C for two times, and the supernatants were collected. Protein concentrations were determined by a Bradford protein assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE, and transferred onto a polyvinylidene fluoride filter (PVDF) membrane (Millipore). Membranes were blocked with 5% non-fat milk, and incubated at 4 °C overnight with anti-NG2, anti-GalC, anti-GDNF, anti-NT-3, anti-BDNF, anti-iNOS, anti-Arginase-1 and anti-β-actin (Cell signaling, Davers, MA, USA). Bands were visualized by HRP-conjugated secondary antibodies (Thermo Scientific, MA, USA) and chemiluminescence (ECL) kit under ECL system (Millipore).
4
Signaling Pathway Antibodies and Assays
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Anti–phosphorylation (p) inhibitor of B (IκB)-α, anti-PKG, anti-VASP, and anti–p-VASP (Ser 239), anti-Arginase1, anti-STAT6, anti–p-STAT6 (Tyr 641), and β-actin antibodies were from Cell Signaling Technology (Beverly, MA). Anti-eNOS mouse polyclonal antibody was obtained from BD Biosciences (Lexington, KY). Anti-GAPDH rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Biosource total Akt and p-Akt (Ser 473) ELISA kits were purchased from Invitrogen (Carlsbad, CA), and IRS2 and p-IRS2 ELISA kits were purchased from Cell Signaling Technology. Lactate was measured using an Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen). (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-IM1,2-diolate (DETA-NO) was purchased from Enzo Life Sciences. DETA-NO was used within 24 h after the reconstitution.
5
Comprehensive Antibody Panel for Research
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anti-FLAG M2 (Sigma, F1804), anti-HA.11 (BioLegend, 901501), anti-Myc (clone 9E10, Santa Cruz Biotechnology, sc-40), anti-GST (Yeasen, 30902ES60), anti-NRP1 (Cell Signaling Technology, #3725), anti-NRP2 (Cell Signaling Technology, #3366), anti-EGLN3 (Novus, NB100–303), anti-Erk3 (Santa Cruz Biotechnology, sc-365234), anti-arginase1 (Cell Signaling Technology, #9819), anti-α-tubulin (Sigma, T6074), anti-β-actin (Yeasen, 30101ES60), anti-GAPDH (Immunoway, YM3029), anti-CD31 (Abcam, ab28364), anti-Mac3 (BD Phmarmingen, 550292), anti-CD8 (InVitrogen, #14–0081–82), anti-F4/80 (Abcam, ab6640), anti-CD25 (Servicebrio, GB11612), anti-VEGF (Novus Biologicals, NB100–664), anti-PCNA (Servicebrio, GB11010), anti-cleaved caspase-3 (Cell Signaling Technology, #9661), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-MMP2 (Abcam, ab37150), anti-phospho-IкBα (Cell Signaling Technology, #2859), anti-iNOS (Cell Signaling Technology, #2982), anti-p53 (Santa Cruz Biotechnology, sc-126), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822), anti-p16 (Santa Cruz Biotechnology, sc-1661), anti-p21 (Santa Cruz Biotechnology, sc-817), normal mouse IgG1 (Santa Cruz Biotechnology, sc-3877), and anti-mouse IgG(H + L) (Jackson ImmunoResearch, 715-585-151).
6
MPTP-Induced Parkinson's Disease Model
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1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 3,4-Dihydroxyphenylacetic acid (DOPAC) were provided by Sigma-Aldrich (Saint Louis, USA). Dopamine (DA) and Homovanillic acid (HVA) were provided by Solarbio (Beijing, China). Anti-Tyrosine Hydroxylase antibody was provided by Abcam (Cambridge, UK), anti-MHC class II was provided by Santa Cruz (Dallas, USA), anti-Arginase-1 was purchased from Cell Signaling Technology (Danvers, MA, USA), goat anti-rabbit IgG and goat anti-mouse IgG conjugated to HRP were purchased from Beyotime Institute of Biotechnology (Shanghai, China), and ELISA Kits were provided by R&D (Minneapolis, USA).
7
Western Blot Analysis of Macrophage Proteins
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Macrophages were lysed in RIPA buffer containing protease inhibitor on the ice for 10 minutes and then centrifuged at 14,000g for 5 minutes to generate protein lysates. The protein concentration was determined by BCA assays and then mixed with 4× Laemmli sample buffer and heated at 95°C for 5 minutes. Protein was separated by 4%–20% gradient SDS-PAGE and transferred onto nitrocellulose membranes. Then, the membranes were blocked with 5% nonfat milk in TBST and immunostained with primary antibodies, anti-GPX4 (Novus, MAB5457, 1:1000) anti-ferritin (Abcam, ab65080, 1:3000), anti-malondialdehyde (Abcam, ab6463, 1:2000), anti-arginase 1 (Cell Signaling Technology, 93668s, 1:1000), and β-actin (Cell Signaling Technology, 4970s, 1:5000) at 4°C overnight and detected using HRP-conjugated secondary antibodies.
8
Immunofluorescence Analysis of Macrophage Markers
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Tissue sections were de‐paraffined, and then heated in citric acid buffer (pH 6.0) at 121°C for 15 min for antigen retrieval. The sections were blocked with 10% bovine serum before incubation with primary antibodies overnight at 4°C: anti‐CD68 (rat monoclonal, dilution 1/100, MCA1957; Bio‐Rad), anti‐IL1RL2 (rabbit polyclonal, dilution 1:200, # PA5‐38013; Invitrogen), anti‐TNF‐α (rabbit monoclonal, dilution 1:400, #11948; Cell Signaling Technology), anti‐iNOS (rabbit monoclonal, dilution 1:400, #13120; Cell Signaling Technology), anti‐Arginase‐1 (rabbit monoclonal, dilution 1:400, #93668; Cell Signaling Technology), and anti‐CD206 (rabbit polyclonal, dilution 0.1 μg/ml, ab64693; Abcam). After incubation, the sections were incubated with Alexa Fluor 488‐conjugated (A11006, 5 μg/ml; Invitrogen) or Alexa Fluor 594‐conjugated (A11012, 5 μg/ml, Invitrogen) secondary antibodies for 1 h, and then mounted with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 10 min at room temperature. Cells were observed using a fluorescence microscope (Biorevo BZ‐90000; Keyence).
9
Comprehensive Immunoblotting Methodology
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Immunoblotting was conducted as described previously [15 (link), 16 (link)]. Primary antibodies applied in this study include the following: anti-NLRP3 (AdipoGen, AG-20B-0014-C100), anti-ASC (AdipoGen, AG-25B-0006), anti-caspase 1 (AdipoGen, AG-20B-0042-C100), anti-interleukin-1β (R&D Systems, AF-401-NA), anti-FLAG (Sigma, F1804), anti-HA (Biolegend, 901501), anti-myc (Santa Cruz Biotechnology, sc-40), anti-gasdermin D (Abcam, ab209845), anti-ABCA1 (Abcam, ab18180), anti-CD36 (Novus Biologicals, NB400-144), anti-Parkin (Cell Signaling Technology, #4211), anti-phosphorylated Parkin Serine 65 (Affinity, AF3500), anti-VDAC1 (Cell Signaling Technology, #4661), anti-LC3 (Sigma, L8918), anti-p62 (MBL, PM045), anti-caspase 8 (Novus Biologicals, NB100-56116), anti-cleaved caspase 3 (Cell Signaling Technology, #9661), anti-arginase 1 (Cell Signaling Technology, #9819), anti-PARP (Cell Signaling Technology, #9542), anti-α-tubulin (Antgene, Wuhan, China; ANT328), anti-β-actin (Yeasen, Shanghai, China; 30101ES50), and anti-GAPDH (Antgene, Wuhan, China; ANT324).
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