Part of the selected n = 188 tumor samples could not be analyzed due to insufficient tumor material to obtain at least one microscopic image (n = 26). Triple immunofluorescent staining for CD3 (ab828, Abcam, Cambride, UK), FoxP3 (ab20034, Abcam) and IL-17 (AF-317-NA, R&D Systems, Abingdon, UK) was performed on 162 tumor samples as described before [29 (link)]. These comprised 86 p16 positive and 76 p16 negative tumors. Images were obtained using an LSM700 confocal laser scanning microscope containing an LCI Plan-Neofluar 25×/0.8 Imm Korr DIC M27 objective (Zeiss, Göttingen, Germany). One to four random images sampled a total vital tumor (epithelium + stroma) area of up to 1.0 mm2. Total tumor epithelium and stroma surface area and double or triple positivity of cells were determined in each image using LSM Image Browser (version 4.2.0.121, Zeiss). Single-, double- and triple-positive cells were scored separately in the tumor epithelial and stromal areas using ImageJ version 1.47 (http://rsb.info.nih.gov/ij ). Cells within blood vessels and largely autofluorescent areas were not scored.
Af 317 na
Manufactured by R&D Systems
Sourced in United States, United Kingdom
AF-317-NA is a recombinant human Wnt-3a protein. It is produced in a mouse myeloma cell line and is purified by proprietary chromatographic techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab828, by Abcam (2 mentions)
Ab20034, by Abcam (2 mentions)
Ls a3391, by LifeSpan BioSciences (2 mentions)
Lsm image browser, by Zeiss (1 mentions)
Lci plan neofluar 25 0.8 imm korr dic m27 objective, by Zeiss (1 mentions)
Streptavidin peroxidase, by Agilent Technologies (1 mentions)
3 amino 9 ethylcarbazole, by Agilent Technologies (1 mentions)
Epitope retrieval solution, by Agilent Technologies (1 mentions)
Protein block serum free solution, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Faramount aqueous mounting medium, by Agilent Technologies (1 mentions)
Clone 195314, by R&D Systems (1 mentions)
Ab68672, by Abcam (1 mentions)
Clone l26, by Agilent Technologies (1 mentions)
Citrate buffer, by Merck Group (1 mentions)
Hematoxylin solution, by Agilent Technologies (1 mentions)
Edta buffer, by Agilent Technologies (1 mentions)
Kp 1 clone, by Agilent Technologies (1 mentions)
Mach 1, by Biocare Medical (1 mentions)
Diaminobenzidine tetrahydrochloride, by Biocare Medical (1 mentions)
12 protocols using af 317 na
1
Quantifying T-Cell Populations in Tumor Samples
2
Immunohistochemical Analysis of IL-17A
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5 µm sections of paraffin embedded skin samples were air-dried overnight at 37 °C, dewaxed and rehydrated. Stainings were performed by an automated BOND system (Leica) according to the manufacturer’s instructions: epitope retrieval was performed at pH6 in epitope retrieval solution (DAKO) and incubated with goat anti-human IL-17A (#AF-317-NA, R&D Systems) followed by a biotinylated anti-goat secondary antibody (#BA-9500-1.5, Vector Laboratories). For detection of specific binding, streptavidin peroxidase and its substrate 3-amino-9-ethyl-carbazole (DAKO) were used. All slides were counter stained with hematoxylin. Stainings without primary antibodies were used as negative control. Positive cells were counted in four to nine visual fields per condition.
3
Comprehensive Immunohistochemical Profiling of FFPE Tumor Samples
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Immunohistochemistry was performed on 4 μm tissue sections from FFPE tumor samples. The slides were dried for 20 min, then deparaffinized in Xylene and rehydrated in ethanol to water. Endogenous peroxidase was blocked for 20 min with 1% H2O2. The slides were then washed, and epitope retrieval was performed using Target Retrieval Solution (DAKO) in 95 °C in a pressure cooker heater for 20 min. Slides were blocked in Protein Block Serum-Free Solution (X0909, DAKO) and later incubated with a primary antibody diluted in PBS containing 5% normal goat serum. Following incubation with primary antibody slides were washed in PBS and incubated with a secondary antibody. Labeled-chromogen substrate solution with Liquid DAB chromogen was applied. The slides were counterstained in Mayer htx before rehydrated and mounted with Faramount Aqueous Mounting Medium (S3025, DAKO).
The following primary antibodies were used: anti-CD68 (M0814, clone KP1, DAKO), anti-CD3 (ab16669, clone SP7, Abcam), anti-CD8 (M710301-2, clone C8/144B, Agilent), anti-FOXP3 (ab20034, clone 236A/E7, Abcam), anti-IL17 (AF-317-NA, R&D Systems), anti-NE (ab68672, Abcam), anti-NKp46 (clone 195314, R&D Systems) and anti-CD1a (EP3622, Ventana).
The following primary antibodies were used: anti-CD68 (M0814, clone KP1, DAKO), anti-CD3 (ab16669, clone SP7, Abcam), anti-CD8 (M710301-2, clone C8/144B, Agilent), anti-FOXP3 (ab20034, clone 236A/E7, Abcam), anti-IL17 (AF-317-NA, R&D Systems), anti-NE (ab68672, Abcam), anti-NKp46 (clone 195314, R&D Systems) and anti-CD1a (EP3622, Ventana).
4
Immunohistochemistry Protocol for Tissue Sections
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2 μm-thick consecutive tissue sections were prepared from formalin-fixed and paraffin-embedded tissues, provided by the Pathology Department of the Humanitas Clinical and Research Center, and processed for immunohistochemistry. Briefly, after deparaffinization and rehydration, antigen retrieval was performed by heat treatment using EDTA buffer (0.25 mM, pH8, Dako) or citrate buffer (0.01 M, pH6, SIGMA-ALDRICH) in water bath at 98 °C for 20 min or pressure cooker. Endogenous peroxidases were blocked by incubation with 3% H2O2 for 15 min at room temperature, followed by incubation for 30 min with 2% BSA to block non-specific binding. The sections were then incubated with primary antibodies anti-human CD68 (Dako, KP-1 clone, diluted 1:1000), CD20 (Dako, L26 clone, diluted 1:200), CD8 (Dako, C8/144B clone, diluted 1:100), PD-1 (Abcam, NAT105 clone, diluted 1:50), CD45RO (Dako, UCHL1 clone, diluted 1:200), IL-17 (R&D, AF-317-NA, 1:500) for 1 h at room temperature, followed by incubation with the detection system MACH 1 (Biocare Medical) or Anti-Goat Polymer kit (Biocare Medical). Diaminobenzidine tetrahydrochloride (Biocare Medical) was used as chromogen. Nuclei were lightly counterstained with a freshly made hematoxylin solution (Dako). The sections were then washed in water, mounted and analysed under an optical microscopy.
5
Immunohistochemical Staining Panel for Cell Markers
6
Immunohistochemical Analysis of Muscle Immune Cells
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CD4+, FOXP3+, and IL-17+ cells were detected in muscle biopsy samples by immunohistochemistry. Acetone-fixed, frozen sections were exposed to monoclonal antibodies against CD4 (Dako clone MT-310), FoxP3 (eBioscience, clone 236A/E7), or IL-17 (R&D systems Clone AF-317-NA) overnight at 4°C. Positive staining was revealed by peroxidase reaction (Dako real™ Detection System (K5001; Dako)). Double immunofluorescence was used to analyse FoxP3+ and CD4+ cell colocalisation. Detection of mouse monoclonal anti-FoxP3 and rabbit polyclonal anti-CD4 (Abcam®) antibodies was performed using Alexa Fluor (AF) 555 goat anti-mouse IgG (L+H) and AF 488 goat anti-rabbit IgG (L+H) secondary antibodies. For control purposes, a mouse IgG1 isotype control was included in the protocol. Stained muscle sections were analyzed with a Leica TCS-SP (UV) confocal microscope at the MSSM-Microscopy Shared Resource Facility (Pitie-Salpetriere Hospital, Paris).
7
Immunohistochemical Analysis of IL-17A in Airway Stenosis
8
Multipotent C2C12 Cell Differentiation
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The C2C12 (mouse mesenchymal precursor) cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in 5% CO2 using Dulbecco's Modified Eagle's Medium (WelGENE, Gyeongsan, Republic of Korea) supplemented with 10% heat-inactivated fetal bovine serum (WelGENE), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Gaithersburg, MD, USA).
C2C12 cells were grown under 5% CO2/20% O2 conditions. C2C12 is a multipotent progenitor cell line that has the ability to transdifferentiate into osteoblast. Cells were seeded in 12-well plates at a density of 1 × 104 cells/well. C2C12 cells were then stimulated in fresh medium (supplemented with 10% FBS) containing nasal tissue extract (20 μg/ml) for 48 h. To neutralise IL-13 or IL-17A, cells were cultured in medium containing neutralizing antibody against IL-13 (0.5 μg/ml, AHC0132, Thermo Scientific) and IL-17A (0.5 μg/ml, AF-317-NA, R&D systems). An irrelevant isotype antibody was used as a negative control. C2C12 cells were treated with 100 ng/ml bone morphogenetic protein-2 (BMP-2) used as a positive control.
C2C12 cells were grown under 5% CO2/20% O2 conditions. C2C12 is a multipotent progenitor cell line that has the ability to transdifferentiate into osteoblast. Cells were seeded in 12-well plates at a density of 1 × 104 cells/well. C2C12 cells were then stimulated in fresh medium (supplemented with 10% FBS) containing nasal tissue extract (20 μg/ml) for 48 h. To neutralise IL-13 or IL-17A, cells were cultured in medium containing neutralizing antibody against IL-13 (0.5 μg/ml, AHC0132, Thermo Scientific) and IL-17A (0.5 μg/ml, AF-317-NA, R&D systems). An irrelevant isotype antibody was used as a negative control. C2C12 cells were treated with 100 ng/ml bone morphogenetic protein-2 (BMP-2) used as a positive control.
9
Multiplex Immunofluorescence Staining of FFPE Tissues
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Triple immunofluorescent staining was performed on 4-μm-thick FFPE sections. After antigen retrieval using Tris–ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris plus 1 mM EDTA pH 9.0), rabbit anti-CD3 (ab828, Abcam, Cambride, UK), mouse IgG1 anti-FoxP3 (ab20034, Abcam) and goat anti-IL-17 (AF-317-NA, R&D Systems, Abingdon, UK) diluted in 1 % w/v bovine serum albumin (BSA) in phosphate-buffered saline (PBS) were incubated at room temperature overnight. Alexa Fluor labeled donkey anti-rabbit-A546 (A10040), donkey anti-mouse-A647 (A31570) and donkey anti-goat-A488 (A11055; all from Invitrogen, Life Technologies, Carlsbad, USA) were incubated at room temperature for 1 h. Slides were mounted using VectaShield mounting medium containing DAPI (Vector Laboratories, Burlingame, USA). For negative controls, the primary antibodies were omitted and substituted with antibodies of the same isotype class with an unknown specificity.
10
Immunohistochemical Staining Panel for Cell Markers
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The following panel of commercially available primary anti-human Abs was included in this study: mouse MC tryptase (clone AA1, M7052, Dako, Glostrup DK; dilution for IHC 1:500, incubation 1 h at 37°C); mouse MC tryptase (clone AA1, ab2378, Abcam, Cambrige UK; dilution for IF 1:600, incubation 2 hr at 37°C); mouse MC chymase (clone CC1, MCCXMS, Aczon Biotech, Bologna, IT; dilution for IHC 1:50, incubation 1 h at 37°C); mouse AhR (clone RPT1, GTX22770, GeneTex, Irvine CA, USA; dilution for IHC 1:100, incubation 16 hr at 4°C); rabbit AhR (LS-A3391, LifeSpan BioSciences, Seattle WA USA; dilution for IF 1:30, incubation 16 hr at 4°C); rabbit IDO1 (clone M80, sc-25809, Santa Cruz Biotechnology, Dallas TX, USA; dilution for IHC 1:400, incubation 1 h at 37°C); goat IL-17 (AF-317-NA, R&D System, Minneapolis MN, USA; dilution for IF 1:25, incubation 3 hr at 37°C); rat IL-10 (clone JES3-9D7, 14-7108, eBiosciences, San Diego CA, USA; dilution for IF 1:80, incubation 3 hr at 37°C).
Secondary conjugated Abs for IF were: anti-mouse Alexa Fluor® 488 (Abcam; dilution 1:800), anti-rabbit 649 (dilution 1:400) and anti-rat DyLight 549 (dilution 1:800), both from Jackson Immunoresearch Europe Ltd. (Suffolk UK), and anti-goat Alexa Fluor® 555 (Invitrogen Thermo Fisher Scientific Inc. Waltham, MA USA; dilution 1:600). Slides were incubated with the fluorescent-labeled secondary Abs for 1 h at 37°C.
Secondary conjugated Abs for IF were: anti-mouse Alexa Fluor® 488 (Abcam; dilution 1:800), anti-rabbit 649 (dilution 1:400) and anti-rat DyLight 549 (dilution 1:800), both from Jackson Immunoresearch Europe Ltd. (Suffolk UK), and anti-goat Alexa Fluor® 555 (Invitrogen Thermo Fisher Scientific Inc. Waltham, MA USA; dilution 1:600). Slides were incubated with the fluorescent-labeled secondary Abs for 1 h at 37°C.
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