Cd31 clone jc70a

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CD31 (clone JC70A) is a laboratory reagent used for the identification and characterization of cells expressing the CD31 antigen, also known as platelet endothelial cell adhesion molecule (PECAM-1). This antibody clone is typically used in immunohistochemistry and flow cytometry applications to detect the presence of CD31-positive cells.

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JC70A (32 citations) Clone JC70A (20 citations) Anti-CD31 (clone JC70A, (9 citations) CD31 (JC70A), (3 citations) Mouse anti-human CD31 antibody (clone JC70A) (3 citations)

21 protocols using cd31 clone jc70a

Immunohistochemical Analysis of Vascular Markers

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Individual cross-sections were incubated with antibodies against aquaporin-1 (clone 1/A5F6, Abcam, Cambridge, UK), ICAM-1 (clone My13, Zymed, South San Francisco, CA), CD31 (clone JC70A, Dako, Glostrup, Denmark), monocyte/macrophage (clone HAM56, Dako), actin (smooth muscle)(clone 1A4, Dako). All antibodies were diluted in TBS containing 1% weight/volume (w/v) bovine serum albumin (BSA), (Sigma-Aldrich, Zwijndrecht, the Netherlands) and 0.01% (w/v) Tween-20 (Sigma-Aldrich). The incubations with secondary biotin-conjugated antibodies (Dako) were followed by amplification with a streptavidin-HRP complex (Dako), and a peroxidase-substrate staining (Nova Red kit, Vector Labs, Burlingame, CA).

Fontijn R.D., Volger O.L., van der Pouw-Kraan T.C., Doddaballapur A., Leyen T., Baggen J.M., Boon R.A, & Horrevoets A.J. (2015). Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo. PLoS ONE, 10(12), e0145777.

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Immunohistochemical Analysis of Signaling Pathways

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Resected tissue samples were fixed with 10% formalin, routinely embedded in paraffin, cut into 4 μm thick serial sections, and used for H&E and immunohistochemical staining. Immunohistochemical staining was performed using a Roche Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. Primary antibodies against p-AKT (#4060, 1:100; Cell Signaling Technology, Danvers, MA, USA), p-mTOR (clone 49F9, 1:100; Cell Signaling Technology), p-S6K1 (#9204, 1:100; Cell Signaling Technology), p-4EBP1 (clone 236B4, 1:500; Cell Signaling Technology), S100 (polyclonal, Ventana Medical Systems), CD31 (clone JC70A, 1:200, Dako), CD34 (clone QBEnd10, 1:200, Dako), D2-40 (760-4395, Ventana Medical System), and PROX1 (ab199359, 1:500; Abcam, Cambridge, UK) were used. Samples were considered positive when at least 10% of the endothelial cells of abnormal vessels exhibited a signal for the targeted protein.

Hori Y., Hirose K., Ozeki M., Hata K., Motooka D., Tahara S., Matsui T., Kohara M., Higashihara H., Ono Y., Tanaka K., Toyosawa S, & Morii E. (2022). PIK3CA mutation correlates with mTOR pathway expression but not clinical and pathological features in Fibfibroipose vascular anomaly (FAVA). Diagnostic Pathology, 17, 19.

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Immunohistochemical Marker Expression Analysis

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Expression intensities of CXCR4, CD31, CD44 and CD81 were carried out on TMAs, whereas one or two tissue cores 1 mm in diameter were analyzed for each sample. TMA sections were deparaffinized and subjected to heat-induced epitope retrieval before incubation with primary antibodies using the automated Leica Bond Max™ system (Leica Microsystems, Wetzlar, Germany). Antibodies were directed against CXCR4 (polyclonal # PA3-305; Thermo Fisher Scientific, Waltham, MA, USA), CD31 (clone JC70A, Dako, Glostrup, Denmark), CD44 (clone DF14/85, Dako, Glostrup, Denmark) and CD81 (clone M38, Thermo Fisher Scientific, Waltham, MA, USA). The detection kit Bond Polymer Refine (Leica Microsystems, Nußloch, Germany) was used for visualization of bound antibodies. Tonsil tissue was used as a positive control. Staining intensity was semi-quantitatively assessed as negative vs. weak, moderate, and strong.

Janjetovic S., Lohneis P., Nogai A., Balci D., Rasche L., Jähne D., Bokemeyer C., Schilling G., Blau I.W, & Schmidt-Hieber M. (2021). Clinical and Biological Characteristics of Medullary and Extramedullary Plasma Cell Dyscrasias. Biology, 10(7), 629.

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Histological Evaluation of Wound Tissue

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The obtained slough was quickly fixed in 10% neutral buffered formalin solution, embedded in paraffin, and cut into 4-μm-thick sections perpendicular to the surface. Hematoxylin/eosin (H/E) staining and Azan–Mallory (A/M) staining were performed. Immunostaining was performed to identify the following cell types: CD33 (clone PWS44, 1×, Leica) for neutrophils, CD68 (clone KP1, 1×, DAKO) for macrophages, CD31 (clone JC70A, 1×, DAKO) for endothelial cells, vimentin (clone V9, 1×, Ventana) for mesenchymal cells, and α-smooth muscle actin (αSMA; clone 1A4, 1×, DAKO) for smooth muscle cells.

Kobayashi H., Imai Y., Hirao T., Nakao K., Kajinaka H, & Kishi K. (2021). Histopathological Analysis of Decellularized Porcine Small Intestinal Submucosa after Treatment of Skin Ulcer. Plastic and Reconstructive Surgery Global Open, 9(12), e3967.

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Seeding Primary Pancreatic Endothelial Cells in Acellular Scaffold

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Human primary pancreatic endothelial cells (hPPECs) were isolated from discarded pancreata as described by Navone et al.^{23 (link)} Cells were then expanded on matrigel–coated flasks and grew with endothelial proliferation medium (EndoPM). At the time of seeding, 20×10⁶ cells were injected with a syringe pump in a whole acellular pancreas scaffold through the SMA and SA, at a flow of 0.2ml/min. Cells were allowed to attach for 2 hours, after which perfusion culture started. Seeded matrices were cultured in a custom-built bioreactor (Fig. 8B). Media were infused through Luer lock access ports and allowed to equilibrate with 5%CO₂ and 95% room air by insertion of 0.22µm filters and magnetic stirring. The system (and the related technical support) was designed and developed by SKE Advanced Therapies.
After 6 days, matrices were fixed in formalin, sampled into 5-µm sections, and stained for H&E, CD31 (clone JC70A, 1:100, DAKO), Ki67 (clone Mib1, 1:600, DAKO) using standard protocols. Antigen retrieval was performed with Tris EDTA pH 9 for both antibodies. The percentage of Ki67+ cells was obtained as the ratio of Ki67+ and hematoxylin+ cells. Immunohistochemistry and H&E-stained images were recorded using Aperio Scan System (Leica Microsystems).

Peloso A., Urbani L., Cravedi P., Katari R., Maghsoudlou P., Alvarez Fallas M.E., Sordi V., Citro A., Purroy C., Niu G., McQuilling J.P., Sittadjody S., Farney A.C., Iskandar S.S., Rogers J., Stratta R.J., Opara E.C., Piemonti L., Furdui C., Soker S., De Coppi P, & Orlando G. (2016). THE HUMAN PANCREAS AS A SOURCE OF PRO-TOLEROGENIC EXTRACELLULAR MATRIX SCAFFOLD FOR A NEW GENERATION BIO-ARTIFICIAL ENDOCRINE PANCREAS. Annals of surgery, 264(1), 169-179.

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Comprehensive Immunohistochemistry Panel for Tumor Diagnosis

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Cases were identified in the consultation files of the authors. The tissue specimens were fixed in formalin and processed routinely for histopathology. Immunohistochemistry (IHC) was performed on 3-μm sections cut from paraffin blocks using a fully automated system (“Benchmark XT System”, Ventana Medical Systems Inc., 1910 Innovation Park Drive, Tucson, Arizona, USA) and the following antibodies: vimentin (V9, 1:100, Dako), keratin cocktail (clone AE1/AE3, 1:40, Zytomed, Berlin, Germany), p63 (SSI6, 1: 100, DCS), desmin (clone D33, 1:250, Dako), alpha smooth muscle actin (clone 1A4, 1:200, Dako), HMB45 (clone HMB45, 1:50, Enzo), Melan A (clone A103, 1:50, Dako), CD34 (clone BI-3C5, 1:200, Zytomed), ERG (EPR3864, prediluted, Ventana), CD31 (clone JC70A, 1:20, Dako), S100 protein (polyclonal, 1:2500, Dako), SOX10 (polyclonal, 1:25, DCS), PAX8 (polyclonal rabbit anti-PAX8, 1:50, Cell Marque), NSE (clone BBS/NC/VI-H1, 1:300, Dako), TTF1 (clone 8G7G3/1, 1:500, Zytomed Systems, Berlin, Germany), Napsin A (MRQ-60, ready-to-use, Medac), calretinin (polyclonal, 1:100, Zytomed), alpha-inhibin (clone R1, 1:50, Serotec), GFAP (Clone GFA, 1/1000, DakoPatts, Denmark), EMA (clone E29, 1:200, Dako), STAT6 (clone S-20, 1:1000, Santa Cruz Biotechnology), TFE3 (clone MRQ-37, 1:100, Cell Marque), Cathepsin-K (clone 3F9, 1:50, Abcam) and Ki67 (clone MiB1, 1:100, Dako).

Agaimy A., Stoehr R., Michal M., Christopoulos P., Winter H., Zhang L., Stenzinger A., Michal M., Mechtersheimer G, & Antonescu C.R. (2021). Recurrent YAP1-TFE3 Gene Fusions in Clear Cell Stromal Tumor of The Lung. The American journal of surgical pathology, 45(11), 1541-1549.

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Immunohistochemical Identification of TMEM Doorways

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As described previously [7 (link)], TMEM doorways are defined as the microanatomical sites of a Mena-overexpressing tumor cell in direct contact with a macrophage and an endothelial cell. As such, FFPE tissues are stained with a sequential triple immunostain for these cell types in the following order:

Endothelial cells—CD-31 (clone JC70A; 1:800 dilution; DAKO, Santa Clara, CA, USA) with Bond Epitope Retrieval Solution 2 and Vector Blue chromogen,

Macrophages—CD-68 (clone PG-M1; 1:300 dilution; DAKO) with antigen retrieval using Bond Epitope Retrieval Solution 1 and 3,3’-Diaminobenzidine (DAB) chromogen,

Tumor cells—anti-pan-Mena antibody (P/N: 610692, BD Biosciences, San Jose, CA, USA) that stain all isoforms of Mena, macrophages with Fast Red chromogen (Bond Polymer Refine Red Detection, Leica Biosystems, Buffalo Grove, IL, USA).

A light green counterstain was also applied to add normal tissue context. All staining was performed on the Bond Max Auto-stainer.

Entenberg D., Oktay M.H., D’Alfonso T., Ginter P.S., Robinson B.D., Xue X., Rohan T.E., Sparano J.A., Jones J.G, & Condeelis J.S. (2020). Validation of an Automated Quantitative Digital Pathology Approach for Scoring TMEM: A Prognostic Biomarker for Metastasis. Cancers, 12(4), 846.

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Peptide-Based Targeting of EGFR in HNSCC

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Disruptin, Biotin-NH-YGRKKRRQRRRSVDNPHVC-CO₂H, NB-Disruptin (non-biotinylated), NH₂-YGRKKRRQRRRSVDNPHVC-CO₂H, RI-Disruptin (retroinverso), NH₂-cvhpndvsrrrqrrkkrgy-CO₂H, and Scram-peptide, Biotin-NH-YGRKKRRQRRRNHVPSDVC-CO₂H were synthesized by SynPeptide Co Ltd. EGFR antibody (cat#4267) was acquired from Cell Signaling Technology, Inc. (Danvers, MA). Ki-67 antibody (ab15580) was acquired from Abcam (Cambridge, MA). CD-31 (clone JC70A) was purchased from (Dako, Carpinteria, CA). The human head and neck squamous cell carcinoma (HNSCC) cell lines, UMSCC11B, and UMSCC47 were kindly provided by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI). The lung cancer cell line, NCI-H1975, was provided by J. Engelman (Massachusetts General Hospital, Boston, MA). A549 cells were purchased from the American Type Culture Collection (Manassas, VA). All cell lines were grown in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (Gibco, Waltham, Massachusetts) and 1X Penicillin-Streptomycin-Glutamine (Gibco# 10378016). Other reagents used in this study were Propidium Iodide (Invitrogen # P1304MP), Matrigel (BD Biosciences # 356237), Harris Hematoxylin (Leica # 3801560), Bluing Reagent (Thermo Scientific #7301), Clarifier 1 (Thermo Scientific #7401). ABC-HRP Kit (PK6100), and DAB Substrate Kit (SK-4100) were purchased from Vector Laboratories.

Mehta R.K., Shukla S., Ramanand S.G., Somnay V., Bridges A.J., Lawrence T.S, & Nyati M.K. (2021). Disruptin, a cell-penetrating peptide degrader of EGFR: Cell-Penetrating Peptide in Cancer Therapy. Translational Oncology, 14(8), 101140.

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Quantitative Immunohistochemical Assessment of Mast Cells and Blood Vessels

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Mast cells in all cases were then immunohistochemically stained for mouse monoclonal anti-human tryptase (clone 10D11, 1:150 dilution; Leica, Mannheim, Germany), mouse monoclonal anti-human cluster of differentiation (CD)31 (clone JC70A, 1:40 dilution; Dako, Carpinteria, CA, USA), mouse monoclonal anti-human CD34 (clone QBEnd10, 1:250 dilution; Dako) and rabbit polyclonal anti-human CD117 (1:100 dilution; Dako) with an automated immunostainer (Bond™ Max; Leica Biosystems, Melbourne, Australia) (9 (link)).
Blood vessel density was assessed by light microscopy according to the method of Weidner et al (10 (link)) and a score graded on a scale of one to four was assigned: 1, 1–5 microvessels observed; 2, 6–10 microvessels observed; 3, 11–15 microvessels observed; 4, 16–20 microvessels observed.

DONATO G., CONFORTI F., CAMASTRA C., AMMENDOLA M., DONATO A, & RENZULLI A. (2014). The role of mast cell tryptases in cardiac myxoma: Histogenesis and development of a challenging tumor. Oncology Letters, 8(1), 379-383.

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Immunohistochemical Profiling of Cervical Tissues

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Tissue sections from ICSCC and normal cervices samples were stained with WWOX (Upstate, NY, USA), p53 (clone DO7, DAKO), Ki-67 (clone MIB-1, DAKO) and CD31 (clone JC/70A, DAKO) antiserum. Briefly, 4-µm paraffin-embedded sections were dewaxed in xylene and hydrated with graded ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ in water for 10 minutes. Heat-induced antigen retrieval was performed with 1 mM EDTA buffer at pH 8.0 for 30 minutes in a steamer at 96°C.
Primary polyclonal rabbit antiserum was used at 1:100, 1:100, 1:100 and 1:40 dilutions for WWOX, p53, Ki-67 and CD31, respectively, for 18 hours at 4°C. This was followed by incubation with the labeled streptavidin-biotin NovoLink Max Polymer Detection System (Leica Biosystems, Nussloch, Germany). The peroxidase activity was developed with DAB (Sigma, St Louis, MI, USA) with timed monitoring using a positive control sample. The sections were then counterstained with hematoxylin, dehydrated and mounted.

Seabra M.A., Cândido E.B., Vidigal P.V., Lamaita R.M., Rodrigues A.N, & Silva Filho A.L. (2018). Immunohistochemical WWOX Expression and Association with Angiogenesis, p53 Expression, Cell Proliferation and Clinicopathological Parameters in Cervical Cancer. RBGO Gynecology & Obstetrics, 40(2), 79-85.

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