Anti cd31

Manufactured by Dianova

Sourced in Germany, United States

Anti-CD31 is a laboratory reagent used for the identification and characterization of cells expressing the CD31 (platelet endothelial cell adhesion molecule-1 or PECAM-1) antigen. CD31 is a transmembrane glycoprotein that is expressed on the surface of various cell types, including endothelial cells, platelets, and some immune cells. This reagent can be used in various applications, such as flow cytometry and immunohistochemistry, to detect and analyze the presence and distribution of CD31-positive cells.

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Lab products found in correlation

Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions) Anti fsp 1, by Merck Group (1 mentions) Anti tdtomato, by Rockland Immunochemicals (1 mentions) Anti vcam 1, by Cell Signaling Technology (1 mentions) Axioimager microscope, by Zeiss (1 mentions) Target retrieve solution, by Agilent Technologies (1 mentions) Anti ki67, by Novus Biologicals (1 mentions) Lsab system hrp kit, by Agilent Technologies (1 mentions) Matrigel, by BD (1 mentions) Anti cd31, by Agilent Technologies (1 mentions) Anti ki67, by Agilent Technologies (1 mentions) Anti f4 80 clone c1 a3 1, by Abcam (1 mentions) Anti ki67, by Abcam (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Laminin, by Agilent Technologies (1 mentions) Fibronectin, by Abcam (1 mentions) Anti α sma, by Abcam (1 mentions) Elastin, by Abcam (1 mentions) Anti ly6g, by Abcam (1 mentions) Anti ki67, by Cell Signaling Technology (1 mentions)

7 protocols using anti cd31

Immunofluorescence Staining of Mouse Tumor Sections

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Immunofluorescence was performed as previously described^{13 (link),14 (link)}. In brief, mouse tumor sections were de-paraffinized and rehydrated, then subjected to antigen retrieval in Target Retrieve Solution (DAKO; S1699) at 95°C for 20 min. Sections were blocked with 5% horse serum for 1 h at room temperature, incubated with anti-CD31 (1:100; Dianova; DIA-310; for mouse tissues), anti-FSP-1 (1:100; Millipore; 07–2274), anti-tdTomato (1:100; Rockland; 600-401-379) or anti-VCAM-1 (1:100; Cell Signaling Technology; 12367) antibody overnight at 4°C. For cell culture staining, the cells were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 1% Triton X-100 for 5 min. Cells were blocked with 5% horse serum for 1 h, then incubated with Alexa Fluor 488-conjugated phalloidin (1:100; Invitrogen; A12379) for 20 min. Images were acquired using an Axio Imager microscope (Zeiss) equipped with an AxioCam 506 monochrome CCD camera (Zeiss).

Cramton S.E., Schnell N.F., Götz F, & Brückner R. (2000). Identification of a New Repetitive Element in Staphylococcus aureus. Infection and Immunity, 68(4), 2344-2348.

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Subcutaneous Xenograft Tumor Model

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Animal protocols were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Johns Hopkins University Animal Care and Use Committee. Mycoplasma-free U87-MG subclones were injected into 6–8 week-old male SCID mice. 5 × 10⁶ cells were suspended in PBS, mixed with an equal volume of Matrigel (BD Bioscience), and injected subcutaneously into the flank. Tumor size was measured with calipers twice per week. Tumor volume (mm³) was calculated using the following formula: length (mm) × width (mm) × height (mm) × 0.52. Tumors were excised and stored at −80°C for RNA and protein extraction or fixed in 10% formalin for paraffin embedding. For immunohistochemistry, paraffin sections were dewaxed, hydrated, and antigens were retrieved with citrate buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween-20, pH 6.2). Immunohistochemistry was performed with an LSAB+ System HRP Kit (Dako) and anti-CD31 (Dianova) or anti-Ki67 (Novus Biologicals) antibody.

Takano N., Sarfraz Y., Gilkes D.M., Chaturvedi P., Xiang L., Suematsu M., Zagzag D, & Semenza G.L. (2014). Decreased Expression of Cystathionine β-Synthase Promotes Glioma Tumorigenesis. Molecular cancer research : MCR, 12(10), 1398-1406.

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Immunohistochemistry protocol for tissue analysis

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The entire mammary fat pad and a sample of liver was excised from all animals at the end of the treatment period. Tissue was fixed in 10% formalin and paraffin embedded. 5 μm sections from each formalin-fixed paraffin-embedded tissue were stained with haematoxylin and eosin (H&E). Antigen retrieval specific to each antibody was performed as follows: by incubation with trypsin (anti-F4/80); by heating in 0.05% tween in Tris-EDTA buffer (anti-Ki67) or by incubation in antigen retrieval buffer (Dako, anti-CD31) [18 (link)]. Blocking was performed with rabbit or goat serum and slides were incubated with the primary antibody for one hour at room temperature [anti-F4/80 Clone C1:A3-1 (abcam, Cambridge, UK) 1:50 dilution, anti-Ki67 (abcam) 1:1000 dilution] or overnight at 4°C [anti-CD31 (Dianova GmbH, Hamburg, Germany) 1:200 dilution] [18 (link)]. Sections were incubated with secondary antibodies raised to the appropriate species using the Vectastain ABC kit (Vector Laboratories Ltd, Peterborough, UK) followed by DAB chromogenic detection.

Rumney R.M., Coffelt S.B., Neale T.A., Dhayade S., Tozer G.M, & Miller G. (2017). PyMT-Maclow: A novel, inducible, murine model for determining the role of CD68 positive cells in breast tumor development. PLoS ONE, 12(12), e0188591.

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Comprehensive histological analysis of tumor tissue

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Tumor tissue samples were fixed in 10% neutral buffered formalin for 12–24 hours at room temperature. Fixed samples were embedded in paraffin blocks, and 5 μm sections were cut. H&E and all IHC staining procedures were performed by TPSR core at Vanderbilt University Medical Center. H&E staining was used for routine morphological analysis. To study ECM elements, we performed Picrosirius red staining and IHC staining for laminin (DAKO), fibronectin (Abcam) and elastin (Abcam). Macrophages, neutrophils, endothelial cells, and proliferating cells were detected using anti-F4/80 (Novus Biologicals LLC), anti-Ly6G (Abcam), anti-CD31 (Dianova), and anti-Ki67 (Cell Signaling Technology) antibodies, respectively. Fluorescent staining was performed with anti-aSMA (Abcam) with secondary anti-mouse Alexa Flour 488, pSMAD3 (Abcam) with secondary anti-rabbit-Biotin and Streptaviden Alexa Flour 647, F4/80 (Novus Biologicals LLC) with anti-rat Alexa Flour 750, CD73 (R&D System) with anti-sheep Alexa Flour 568. Pictures were taken on Keyence BZ-X710. Whole image scanning was performed on Apiro Versa 200 (Leica).

Vasiukov G., Novitskaya T., Zijlstra A., Owens P., Ye F., Zhao Z., Moses H.L., Blackwell T., Feoktistov I, & Novitskiy S.V. (2020). Myeloid cell-derived TGF-beta signaling regulates ECM deposition in mammary carcinoma via adenosine-dependent mechanisms. Cancer research, 80(12), 2628-2638.

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Dual Immunofluorescence for PDGFRβ and CD31

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For PDGFRβ/CD31 dual immunofluorescence staining, antigen retrieval was performed with Tris/Borate/EDTA buffer, pH 8.0–8.5 (Ventana) for 1 hour at RT. Tissue sections were then incubated with primary anti-PDGFRβ (rabbit monoclonal, clone 28E1; Cell Signaling; 40 μg/ml) and anti-CD31 (rat monoclonal, clone SZ31; Dianova; 0.3 μg/ml) for 1 hour at 37°C. Secondary antibody staining was performed sequentially for the two antigens. First, tissue sections were stained with an OmniMap anti-rabbit HRP and developed with Discovery FITC substrate, followed by a peroxidase inhibitor step to quench residual HRP activity. Next, tissue sections were stained with an OmniMap anti-rat HRP and developed with Discovery Rhodamine substrate. Tissue sections were counterstained with the nuclear DNA stain DAPI and visualized by confocal microscopy.

Cho J.S., Fang T.C., Reynolds T.L., Sofia D.J., Hamann S, & Burkly L.C. (2016). PDGF-BB Promotes Type I IFN-Dependent Vascular Alterations and Monocyte Recruitment in a Model of Dermal Fibrosis. PLoS ONE, 11(9), e0162758.

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Tissue Preparation and Immunohistochemistry

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After euthanasia, mice were subjected to transcardial perfusion with PBS + heparin (Sigma-Aldrich) and 4% paraformaldehyde. Tissue samples were collected and fixed in 4% PFA in 1× PBS overnight and embedded in paraffin. Sections were stained with hematoxylin-eosin for analysis. For immunohistochemistry, antibody detection was performed using the Elite ABC kit and DAB substrate according to the manufacturer's instructions (Vector Laboratories) and counterstained with hematoxylin (Thermo Fisher Scientific). Primary antibodies used were rat anti-FLAG (Biolegend 637319), anti-CD31 (Dianova DIA-310), anti-endomucin (Santa Cruz Biotechnology sc-65495), rabbit anti-S100a6 (Novus Biologicals NBP1-89388), anti-ERG (Abcam ab133264), anti-CD34 (Abcam ab81289), anti-Ki67 (Leica Biosystems NCLKi67p), anti-cytokeratin, wide spectrum screening (Agilent Z0622), anti-CRYAB (Invitrogen PA1-16950), and mouse anti-PDGFA (Santa Cruz Biotechnology sc-9974). Alexa488-, Alexa568-, and Alexa647-conjugated secondary antibodies (Molecular Probes) were used for immunofluorescence.

Driskill J.H., Zheng Y., Wu B.K., Wang L., Cai J., Rakheja D., Dellinger M, & Pan D. (2021). WWTR1(TAZ)-CAMTA1 reprograms endothelial cells to drive epithelioid hemangioendothelioma. Genes & Development, 35(7-8), 495-511.

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Immunohistochemical Analysis of Tumor Samples

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After antigen retrieval and blocking (Supplemental Table S1), tumor sections were incubated overnight at 4°C with anti-pimonidazole (1/400, Hypoxyprobe, Massachusetts, USA), anti-caspase-3 (ready to use, Biocare Medical, Concord CA, USA) or anti-CD31 (1/25, Dianova, Hamburg, Germany), anti-GLI1 (1/50, Santa-Cruz), anti-GLI2 (1/1000, Rockland), anti-PTCH1 (1/300, Santa Cruz); or for 30 min at room temperature with anti-Ki67 (ready to use, Thermo Scientific). Appropriate secondary antibodies followed by 3.3′-diaminobenzidine (DAB) substrate (DAKO, Glostrup, Denmark) were used to visualize antigen presence.
Quantification of Ki67 was performed by counting the number of Ki67 positive nuclei in the tumor tissue. Mean vessel density (MVD) was assessed as the number of blood vessels (CD31⁺) per field for 10 high-power fields per tissue specimen. Tumor hypoxic fraction was determined as the percentage of cytoplasmic pimonidazole positive cells. The apoptotic fraction was determined by assessing the number of caspase-3 positive cells per tissue section.

Gonnissen A., Isebaert S., McKee C.M., Dok R., Haustermans K, & Muschel R.J. (2016). The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in vitro and in vivo. Oncotarget, 7(51), 84286-84298.

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