Clone 206d

Manufactured by BioLegend

Sourced in Denmark

Clone 206D is a mouse monoclonal antibody that recognizes the CD206 antigen. CD206, also known as the mannose receptor, is a type I transmembrane protein expressed on the surface of various cell types, including macrophages and dendritic cells. The core function of Clone 206D is to serve as a reagent for the detection and analysis of CD206-positive cells.

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Lab products found in correlation

Clone c8 144b, by Agilent Technologies (2 mentions) Pd l1 clone e1l3n, by Cell Signaling Technology (1 mentions) Pd 1 clone epr4877 2, by Abcam (1 mentions) Clone ae1 ae3, by Agilent Technologies (1 mentions) Clone mib 1, by Agilent Technologies (1 mentions) Clone pg m1, by Agilent Technologies (1 mentions) Bond max, by Leica Biosystems (1 mentions) Foxp3, by BioLegend (1 mentions) Clone ln10, by Leica camera (1 mentions) Clone kp1, by Agilent Technologies (1 mentions) Ventana benchmark ultra immunostainer, by Roche (1 mentions) Benchmark ultra, by Roche (1 mentions) Clone e1l3n, by Cell Signaling Technology (1 mentions) Benchmark automated stainer, by Roche (1 mentions)

6 protocols using clone 206d

Immunohistochemical Analysis of T-cell Subsets

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Immunohistochemistry for CD4, CD8, and FOXP3 were performed on 4-µm unstained sections from the tissue microarray blocks using primary mouse monoclonal antibodies against CD4 (1:25 dilution, SP35; Cell Marque, Rocklin, Calif), CD8 (1:20 dilution from predilute, C8/144B; Thermo Scientific, Fremont, Calif), and FOXP3 (1:50 dilution, clone 206D, BioLegend, San Diego, Calif). The tissue sections were deparaffinized and placed in a 100°C steamer containing 20 mmol citrate buffer (pH 6.0) for 5 minutes for antigen retrieval. The sections were then incubated with the primary antibody at 35°C for 15 minutes, immersed in 3.0% hydrogen peroxidase at 35°C for 5 minutes to block endogenous peroxide activity, immersed in polymer enhancer for 8 minutes, and then incubated with secondary poly-horseradish peroxidase anti-mouse/anti-rabbit immunoglobulin G at 35°C for 8 minutes. The 3,3′-diaminobenzidine was used as a chromogen. Hematoxylin was used for counterstaining.

Nejati R., Goldstein J.B., Halperin D.M., Wang H., Hejazi N., Rashid A., Katz M.H., Lee J.E., Fleming J.B., Rodriguez-Canales J., Blando J., Wistuba I.I., Maitra A., Wolff R.A., Varadhachary G.R, & Wang H. (2017). Prognostic Significance of Tumor Infiltrating Lymphocytes in Patients With Pancreatic Ductal Adenocarcinoma Treated With Neoadjuvant Chemotherapy. Pancreas, 46(9), 1180-1187.

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Comprehensive Immunohistochemical Analysis

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Chromogen-based IHC analysis was performed by using an automated staining system (BOND-MAX; Leica Biosystems, Vista, CA) with antibodies against the following: pancytokeratin (epithelial cell marker; clone AE1/AE3, dilution 1:300; Dako, Carpinteria, CA), PD-L1 (clone E1L3N, dilution 1:100; Cell Signaling Technology, Danvers, MA), CD8 (cytotoxic T-cell marker; clone C8/144B, dilution 1:20; Thermo Fisher Scientific, Waltham, MA), CD3 (T-cell lymphocyte marker; clone D7A6E, dilution 1:100; Dako), PD-1 (clone EPR4877-2, dilution 1:250; Abcam, Cambridge, MA), Foxp3 (regulatory T-cell marker; clone 206D, dilution 1:50; BioLegend, San Diego, CA), KI67 (proliferation marker; clone MIB-1, dilution 1:100; Agilent Technologies, Santa Clara, CA), and CD68 (macrophage marker; clone PG-M1, dilution 1:450; Dako). Expression of all cell markers was detected using a Novocastra Bond Polymer Refine Detection Kit (catalog #DS9800; Leica Biosystems) with a diaminobenzidine reaction to detect antibody labelling and hematoxylin counterstaining. To guarantee specificity and sensitivity of the different antibodies, several tests were done until we obtained a reproducible pattern and correct geographical distribution of the different antibodies in the control tissues (Supplementary Fig. 1).

Parra E.R., Zhai J., Tamegnon A., Zhou N., Pandurengan R.K., Barreto C., Jiang M., Rice D.C., Creasy C., Vaporciyan A.A., Hofstetter W.L., Tsao A.S., Wistuba I.I., Sepesi B, & Haymaker C. (2021). Identification of distinct immune landscapes using an automated nine-color multiplex immunofluorescence staining panel and image analysis in paraffin tumor tissues. Scientific Reports, 11, 4530.

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Prostate Tumor Immune Infiltrates Characterization

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To characterize prominent prostate tumor infiltrates, we focused on abundant immunologic cell sub-types. We cut, stained and imaged archived formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from the SCORE cases. Then we analyzed their cell type and count in the Department of Pathology and Laboratory Medicine at the University of Pennsylvania. Prostate sections from resected glands were stained for cytokeratin from tumor cells and T cells (CD3, CD8, FoxP3) and macrophages (CD68.) (Fig 1) The antibody clones that we used were CD3: clone LN 10 (Leica, catalog #NCL-L-3); CD8: clone C8/144B (DAKO, catalog #M7103); FOXP3: clone 206D (Biolegend, catalog #320102); and CD68: clone KP1 (DAKO, catalog #IR60961). The entire tumor nodule was scanned at low power to survey overall prevalence of infiltrating immune cells. We then selected 4 representative fields from each tumor sample. The area of the tumor that was selected depended on where the majority of cells were found in each tumor sample. Tissues were scored for infiltrating lymphocytes and macrophages in the dominant nodule (largest tumor nodule identified in the prostate) [21 (link)] by manually counting the number of TILs or TAMs in 4 fields at 20X for T cells and macrophages and averaging the scores.

Zeigler-Johnson C., Morales K.H., Lal P, & Feldman M. (2016). The Relationship between Obesity, Prostate Tumor Infiltrating Lymphocytes and Macrophages, and Biochemical Failure. PLoS ONE, 11(8), e0159109.

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Immunohistochemical Profiling of Tumor Microenvironment

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Immunohistochemical (IHC) staining was performed on 4 μm thick sections of formalin-fixed, paraffin-embedded tissue samples using an automated staining platform (Ventana Benchmark Ultra immunostainer, Ventana Medical Systems, Inc., Tucson, USA).
Immunostaining was performed with anti-CD3 (clone SP7, #RM9107-S1, Thermo Fisher Scientific, Cheshire, UK), anti-CD8 (clone C8/144B, #M7103, Dako, Glostrup, Denmark), anti-CD45RO (clone UCHL1, #M074201, Dako, Glostrup, Denmark) and anti-FOXP3 (clone 206D, #320116, BioLegend, San Diego, CA, USA) antibodies using an autostainer (Benchmark Ultra, Ventana Medical Systems, Tucson, USA) according to the manufacturer’s instructions. In negative controls the primary antibody was omitted. A mediastinal lymph node served as positive control. The presence of lymphatic vessel invasion in PM was known from a previous study [19 (link)].

Schweiger T., Berghoff A.S., Glogner C., Glueck O., Rajky O., Traxler D., Birner P., Preusser M., Klepetko W, & Hoetzenecker K. (2016). Tumor-infiltrating lymphocyte subsets and tertiary lymphoid structures in pulmonary metastases from colorectal cancer. Clinical & Experimental Metastasis, 33(7), 727-739.

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Immunohistochemical Analysis of PDA Tumor Samples

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Study cases were obtained from the surgical pathology files at Thomas Jefferson University with Institutional Review Board approval. The tissue microarray (TMA) contained tumor samples derived from 223 largely consecutive patients with PDA who had been treated at Thomas Jefferson University Hospitals between the years 2002 and 2010. Whole tissue section slides were constructed and TMAs were derived from them using a tissue arrayer (Veridiam) as previously described (4 (link)). Immunohistochemistry was performed as previously described (21 (link)) on 4μm TMA sections using a standard avidin-biotin immunoperoxidase method with antibodies specific for CD163 (1:100, clone 10D6, Leica), FOXP3 (1:50, clone 206D, Biolegend), PD-L1 (1:200, clone E1L3N, Cell Signaling Technologies), and MCT4 (1:250, developed and characterized by Dr. Nancy Philp (22 (link))). Staining was performed on a Ventana Benchmark automated stainer.

Hutcheson J., Balaji U., Porembka M.R., Wachsmann M.B., McCue P.A., Knudsen E.S, & Witkiewicz A.K. (2016). Immunological and Metabolic Features of Pancreatic Ductal Adenocarcinoma Define Prognostic Subtypes of Disease. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(14), 3606-3617.

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ChIP Assay of Jurkat T Cells

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To perform chromatic immunoprecipitation (chIP) of Jurkat T cells and/or primary T cells, we used the Simple ChIP Plus enzymatic chromatin IP kit (Cell Signaling Technology) with the addition of anti-FOXP3 antibody Clone 206D (Biolegend) or the Mouse IgG1 isotype κ (BD) control antibody. DNA eluted from IPs was amplified by PCR (Apex 2.0 × Taq RED mastermix) with SAP promoter primers SAP-F 5′-aagttattcctggtggcctc-3′, and SAP-R 5′-ttggttcgatcgagcttgcc-3′. Cycling conditions were 95°C for 180 seconds, 32 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by 72°C for 5 minutes. PCR products were then electrophoresed on a 1% agarose gel (1× TAE) with ethidium bromide for visualization.

Katz G., Voss K., Yan T.F., Kim Y.C., Kortum R.L., Scott D.W, & Snow A.L. (2018). FOXP3 renders activated human regulatory T cells resistant to restimulation-induced cell death by suppressing SAP expression. Cellular immunology, 327, 54-61.

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