Formalin-fixed, paraffin-embedded skin punch biopsies from CTCL patients were obtained from Memorial Sloan Kettering’s pathology archives. Five (5) μm sections were stained for Arginase −1 (Cell Signaling Technology), GATA-3, Tbet, Bcl6 and Foxp3 (all from Santa-Cruz Biotechnology). Staining was performed with a Bond Max Autostainer at the Molecular Cytology Core and Pathology Core Facilities of Memorial Sloan Kettering Cancer Center (Leica Biosystems, Buffalo Grove, IL, USA). All slides were scanned with a Mirax slide scanner (Zeiss Microscopy, Jena, Germany) and digital images were analyzed with Panoramic viewer software (3DHistech Ltd). Arginase-1 quantification was defined as a percentage of Arginase-1 positive myeloid cells within the entire hematolymphoid cutaneous infiltrate. Arginase-1 positive samples were defined as samples containing at least 1% Arginase-1 positive myeloid cells in the dermis. In regard to malignant T-helper polarization, GATA3, Tbet, Bcl6 and Foxp3 expression was quantified for all four transcription factors as a percent-positive fraction among malignant T cells, which were defined morphologically by their nuclear atypia and features of epidermotropism or folliculotropism.
Arginase 1
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Arginase-1 is an enzyme that catalyzes the conversion of L-arginine to L-ornithine and urea. It is involved in the urea cycle and plays a role in regulating the availability of L-arginine for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
P stat6, by Cell Signaling Technology (2 mentions)
P stat1, by Cell Signaling Technology (2 mentions)
Ab16669, by Thermo Fisher Scientific (2 mentions)
Nanozoomer 2.0 ht, by Meyer Instruments (2 mentions)
Npd view2, by Hamamatsu Photonics (2 mentions)
Cd163, by Abcam (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Stat6, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Tnf α, by Cell Signaling Technology (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Bond max autostainer, by Leica (1 mentions)
Mirax slide scanner, by Zeiss (1 mentions)
T bet, by Santa Cruz Biotechnology (1 mentions)
Gata3, by Santa Cruz Biotechnology (1 mentions)
Panoramic viewer software, by 3DHISTECH (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
28 protocols using arginase 1
1
Comprehensive Immunophenotyping of Cutaneous T-Cell Lymphoma
2
Western Blot Analysis of Inflammation Markers
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Cells were harvested and suspended in lysis buffer containing protease and phosphatase inhibitor cocktails. Nuclear protein extraction was performed using NE-PER® nuclear and cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL, USA). Concentrations of proteins were determined using a BCA protein assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were resolved by 8% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skimmed milk for 1 h at room temperature, and then incubated with specific primary antibodies recognizing COX-2, iNOS, arginase -1, PPAR-γ, and NF-κB p-p65 (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Anti-rabbit horseradish-linked IgG was used as the secondary antibody. Signals were developed using the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
3
Protein Analysis Using Chemiluminescence
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For protein analysis, blots were developed using ECL chemiluminescent detection system (GE Life Science, Buckinghamshire, UK). p-STAT1 (#7649), p-STAT6 (#56554), Arginase 1 (#93668), and β-tubulin (#2128) were purchased from Cell Signaling (MA, USA). CD47 (GTX53912), SIRPα (GTX112645) were purchased from GeneTex (CA, USA). iNOS (ab3523) was purchased from Abcam (Cambridge, UK),
4
Immunohistochemical Analysis of Tumor Samples
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Hematoxylin staining and immunostaining were performed on paraffin sections of tumors. Antibodies used were against LILRB4 (lab produced, 1:100), CD3 (Abcam, ab16669, 1:100), PD-1 (Thermo Fisher, J116, 14–9989-82, 1:100) and Arginase-1 (Cell signaling, 9819S, 1:100). The images were visualized using the Hamamatsu NanoZoomer 2.0-HT (Meyer instruments Inc., Houston, TX) and viewed in NPDview2 software (Hamamatsu, Japan).
5
Western Blot Analysis of Murine Jejunal Proteins
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Mice jejuna were dissected out post euthanasia on days 3, 7 and 21 following treatment. The jejunal tissues were incubated in ice-cold lysis buffer (RIPA) with 1 mM phenyl-methylsulfonyl fluoride (PMSF) and complete protease inhibitor cocktail (Sigma, USA) on ice and homogenized to extract protein. The protein concentrations were quantified using BCA protein assay kit (Thermo Scientific, USA). Samples containing equal amounts of protein (50 μg) were resolved using 8–15% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham) as described earlier33 (link). The membranes were subjected to overnight incubation at 4° C with primary antibody against PCNA, Caspase-3, PARP, NF-κB, p53, p21, Cyclin D1, Cyclin B1, Catalase, SOD-2, acetylated-histone H3 (K9/14), β-actin (Santa Cruz Biotechnology, Inc., USA) and Bcl-2, Bax, phospho-CHK2 and Arginase-1 (Cell Signaling Technologies, USA). The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibody at room temperature. The proteins were detected with a chemiluminescent substrate and signal captured using Michrochemi (DNR Bioimaging Systems, Israel).
6
Quantifying Tumor-Associated Macrophages and Angiogenesis
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Isolated perfused tumors were fixed in buffered 10% formalin and embedded in paraffin to be sliced into horizontal 5 μm sections for immunohistochemical and immunofluorescent staining. To calculate TAM marker-positive structure and CD31-positive area in the tumor, we quantitated at least 4 view fields per tumor by using the Image J program, downloaded from the National Institutes of Health (NIH, Bethesda, MD, USA) website. The antibodies against Iba 1 (Abcam, Cambridge, UK, cat #ab5076), CD11b (BIO-RAD, Hercules, CA, USA, cat #MCA711GT), CD11c (Abcam, Cambridge, UK, cat #ab52632), Arginase 1 (Cell signaling, Danvers, MA, USA, cat # #93668), CD206 (Abcam, Cambridge, UK, cat #ab64693) and CD163 (Abcam, Cambridge, UK, cat #ab182422) were used, and ki67 was purchased from Abcam (Cambridge, UK, cat #ab16667). Anti-Nuclei Antibody, clone 235–1, Cy3 conjugate was purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Histological Analysis of Tumor Samples
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Tumor tissues were fixed with 4% formalin, dehydrated in ethanol, embedded in paraffin, cut on microtome and stained with H&E or antibodies according to standard protocols. Antibodies used in the analysis were detyrosinated tubulin (Abcam), alpha‐tubulin (Merck Millipore), E‐cadherin (Cell Signaling Technology, Danvers, MA, USA), CD3 (Nichirei Biosciences, Tokyo, Japan), CD4 (Cell Signaling Technology), CD8 (Cell Signaling Technology), Ly‐6G (Abcam), CD11b (Abcam), F4/80 (Bio‐Rad), Arginase‐1 (Cell Signaling Technology), and CXCL1 (Abcam). Staining signals were visualized using Histofine Simple Stain MAX PO (Nichirei Biosciences) or SignalStain Boost IHC Detection reagent (Cell Signaling Technology) followed by counterstaining with hematoxylin. For microscopic fluorescence analysis, secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Thermo Fisher Scientific, Waltham, MA, USA) were used. Nuclei were stained with DAPI. The images were captured with DM2000 LED (Leica Microsystems, Wetzlar, Germany) or KEYENCE BZ‐9000 microscope (Keyence Corporation, Osaka, Japan).
8
Polarization of Murine and Human Macrophages
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Polyinosinic–polycytidylic acid (poly(I:C)) was purchased from InvivoGen. Mouse IFNβ (#12405-1) was from PBL. Mouse M-CSF (#315-02), mouse recombinant IL-6 (#315-05), mouse recombinant IL-4 (#214-14), human recombinant M-CSF (#300-25-2), human recombinant IL-6 (#200-06), human recombinant IL-4 (#200-04) were from PeproTech. The ELISA kits for mIL-6 (#88-7064-22) and mIL-4 (#88-7044-22) were from eBioscience.
Mouse IL-4-neutralizing antibodies were from Bio-x cell (#BE0045), mouse IL-6-neutralizing antibody (#504512) was from Biolegend. The ERK1/2 inhibitor U0126 (#U120) was from Sigma. SHP099 (#HY-1003881) was from MCE. The NE-PER Nuclear and Cytoplasmic Extraction kit (#78833) was from Thermo Fisher. Primary antibodies for Western blot were purchased from Cell Signaling Technology (arginase-1, #93668; pSTAT6-Y641, #565543; STAT6, #93623; pSTAT3-Tyr705, #9145; STAT3, #9139; STAT1, #14994; Erk1/2, #4695; pErk1/2, #4370), and Genscript (Actin, #A00730), Abclonal (GFP, #AE012). Antibodies for Flow Cytometry were purchased form Biolegend: anti-CD16/32 (#101330), AF488-anti-Ly6C (#128022), BV421-anti-F4/80 (#123132), AF647-anti-CD206 (#141712).
Mouse IL-4-neutralizing antibodies were from Bio-x cell (#BE0045), mouse IL-6-neutralizing antibody (#504512) was from Biolegend. The ERK1/2 inhibitor U0126 (#U120) was from Sigma. SHP099 (#HY-1003881) was from MCE. The NE-PER Nuclear and Cytoplasmic Extraction kit (#78833) was from Thermo Fisher. Primary antibodies for Western blot were purchased from Cell Signaling Technology (arginase-1, #93668; pSTAT6-Y641, #565543; STAT6, #93623; pSTAT3-Tyr705, #9145; STAT3, #9139; STAT1, #14994; Erk1/2, #4695; pErk1/2, #4370), and Genscript (Actin, #A00730), Abclonal (GFP, #AE012). Antibodies for Flow Cytometry were purchased form Biolegend: anti-CD16/32 (#101330), AF488-anti-Ly6C (#128022), BV421-anti-F4/80 (#123132), AF647-anti-CD206 (#141712).
9
Immunohistochemical Analysis of Inflammatory Markers
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Captured sections were rehydrated and overnight epitope retrieval was performed in sodium citrate buffer at 60°C. Tissue sections were blocked with 5% BSA in TBS containing 0.2% Tween‐20 (TBST) at RT for 1 h and incubated overnight with primary antibodies for iNOS (Abcam; ab15323; rabbit polyclonal; 1:50), Arginase‐1 (Cell Signaling Technologies; #93668; rabbit monoclonal; 1:200), or Ly6G (Invitrogen; #14‐5931‐82; rat monoclonal; 1:100). Secondary antibodies were probed for 2 h at RT using HRP‐(rabbit) or AP‐(rat) conjugates (Jackson Immunolabs). Tissues were developed with ImmPACT DAB (HRP) or Vector Red (AP) substrate (Vector Labs). Arginase‐1 and Ly6G sections were counterstained with hematoxylin while iNOS sections were left without counterstain. Tissues were dehydrated through alcohol and xylene and mounted with Cytoseal XYL. Images were collected on an Olympus BX43 upright microscope equipped with an Olympus DP74 CMOS camera operated by cellSens Standard software. Images were quantified in ImageJ/FIJI.
10
Immunoblotting of Macrophage Markers
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Arginase-1 1:1000 (Cell Signaling Technologies, Beverly, MA, USA). Cathepsin L 1:2000 (R&D Systems, Minneapolis, MN, USA). CD206 1:1000 (ThermoFisher Scientific). Actin 1:20,000 (Sigma-Aldrich, St. Louis, MO, USA)
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