Anti cd31 antibody

Manufactured by BD

Sourced in United States, France

The Anti-CD31 antibody is a laboratory reagent used for the detection and analysis of the CD31 protein, also known as Platelet Endothelial Cell Adhesion Molecule (PECAM-1). CD31 is expressed on the surface of endothelial cells, platelets, and certain leukocytes. The Anti-CD31 antibody can be utilized in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and study cells expressing the CD31 marker.

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Spelling variants of this product

Anti-CD31 (170 citations) CD31 antibody (42 citations) Rat anti-CD31 antibody (20 citations) Rat anti-mouse CD31 monoclonal antibody (14 citations) Monoclonal rat anti-CD31 antibody (8 citations) Rat anti-mouse CD31 primary antibody (6 citations) Monoclonal mouse anti-CD31 antibody (5 citations) Primary anti-CD31 antibody (3 citations) PE)-conjugated mouse anti-human CD31 antibody (2 citations) Anti-CD31-PE antibody (2 citations)

62 protocols using anti cd31 antibody

Immunostaining Protocols for Mouse Microglia and Vasculature

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For mouse microglia immunostaining, eyes were enucleated and fixed in paraformaldehyde 4% for 1 hour. Subsequently, retinas and choroids were isolated, permeabilized, blocked and incubated with anti-Iba1 antibody (1/1000 dilution) (Abcam Ab178846) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rabbit AlexaFluor 595, Invitrogen A11012, USA) for 2 hours and washed again. Retinas and choroids were flat-mounted with Fluoromount-G (SouthernBiotech, The Netherlands) on glass-slides for microscopy imaging.
For mouse vascular immunostaining, eyes were enucleated and fixed in ethanol 70 % for 1 hour. Subsequently, choroids were isolated, permeabilized, blocked and incubated with anti-CD31 antibody (1/150 dilution) (Pharmingen 553370) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rat AlexaFluor 488, Invitrogen A11006) for 2 hours and washed again. Choroids were flat-mounted with Fluoromount-G (SouthernBiotech) on glass-slides for microscopy imaging.

Roblain Q., Louis T., Yip C., Baudin L., Struman I., Caolo V., Lambert V., Lecomte J., Noël A, & Heymans S. (2021). Intravitreal injection of anti-miRs against miR-142-3p reduces angiogenesis and microglia activation in a mouse model of laser-induced choroidal neovascularization. Aging (Albany NY), 13(9), 12359-12377.

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Aortic Ring Assay for Angiogenesis

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To investigate the angiogenic potential of WT and EVL^−/− mice, the aortic ring assay was performed as described previously (Zippel et al, 2016). Briefly, aortas were dissected from mice, cleaned, cut into 1 mm rings, and embedded in collagen drops in a 48‐well plate. The rings were cultivated in microvascular endothelial cell growth medium (PeloBiotech) containing 10 mM l‐glutamine and 2% autologous serum, which was obtained from the same animals and pooled from WT and EVL^−/−. The stimulation was done with 30 ng/ml murine VEGF (PeproTech, #450‐32). After 5 days, the rings were fixed with 4% PFA, immunostained with an anti‐CD31 antibody (BD Pharmingen, #550274) and analyzed using confocal microscopy (Leica, SP8).

Zink J., Frye M., Frömel T., Carlantoni C., John D., Schreier D., Weigert A., Laban H., Salinas G., Stingl H., Günther L., Popp R., Hu J., Vanhollebeke B., Schmidt H., Acker‐Palmer A., Renné T., Fleming I, & Benz P.M. (2021). EVL regulates VEGF receptor‐2 internalization and signaling in developmental angiogenesis. EMBO Reports, 22(2), e48961.

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Quantifying Capillary Density in Wound Healing

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Capillary density in the healing wounds was quantified by immunohistological analysis. Sections were deparaffinized and hydrated, then placed in Tris-Buffered Saline (pH 7.5) for 5 minutes for pH adjustment. Endogenous peroxidase was blocked by 3% hydrogen peroxide/methanol bath for 20 minutes, followed by distilled water rinses. Slides were blocked with normal rabbit serum (Vector Laboratories) for 30 minutes, then incubated for 60 minutes at room temperature with an anti-CD31 antibody (1:50; BD Bioscience) and Vascular endothelial growth factor (VEGF, abcam). Slides were counterstained with Gill 2 Hematoxylin for 10 seconds, differentiated in 1% aqueous glacial acetic acid, and rinsed in running tap water. Twenty random microscopic fields (×200 magnification) were counted to determine the number of capillaries per wound.

Thandavarayan R.A., Garikipati V.N., Joladarashi D., Babu S.S., Jeyabal P., Verma S.K., Mackie A.R., Khan M., Arumugam S., Watanabe K., Kishore R, & Krishnamurthy P. (2015). Sirtuin-6 deficiency exacerbates diabetes induced impairment of wound healing. Experimental dermatology, 24(10), 773-778.

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Immunofluorescence Analysis of Tumor Microenvironment

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Frozen sections of tumor and normal tissues were subjected to immunofluorescence staining. Anti-CD68 antibody (Biorad, CA) and anti-CD163 antibody (Santa Cruz Biotechnology, Inc., CA) were used to identify macrophages. Anti-CD31 antibody (BD Bioscience, NJ) was used to identify endothelial cells in tumor blood vessels, and anti-Ki67 antibody (eBioscience, CA) was used to label proliferating cells. Alexa-Fluor 488 or Alexa-Fluor 555-labeled secondary antibodies (Invitrogen, CA) were used to visualize biomarker positive cells.

Gao N., Bozeman E.N., Qian W., Wang L., Chen H., Lipowska M., Staley C.A., Wang Y.A., Mao H, & Yang L. (2017). Tumor Penetrating Theranostic Nanoparticles for Enhancement of Targeted and Image-guided Drug Delivery into Peritoneal Tumors following Intraperitoneal Delivery. Theranostics, 7(6), 1689-1704.

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Evaluation of Tumor Apoptosis and Angiogenesis

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Frozen tissue sections (6–8 μm thick) of tumors were fixed in cold acetone (4°C) for 10 minutes [13] (link), [20] (link) and processed for immunoflourescent staining. Topro-3 nuclear staining (Life Technologies) was used in conjunction with all immunoflourescent staining to visualize cell nuclei. To evaluate apoptosis and tumor angiogenesis at the border-zone between tumor tissue and surrounding tissue (peri-tumor), sections were triple stained with ApopTag Fluorescein In Situ Apoptosis TUNEL Kit (Millipore, Billerica, MA) anti-CD31 antibody (BD Pharmingen, San Jose, CA) along with Alexa Fluor 555 goat anti-rat secondary (Life Technologies) and Topro-3. The peri-tumoral and tumor area were identified by H&E staining of adjacent sections.

Sasi S.P., Bae S., Song J., Perepletchikov A., Schneider D., Carrozza J., Yan X., Kishore R., Enderling H, & Goukassian D.A. (2014). Therapeutic Non-Toxic Doses of TNF Induce Significant Regression in TNFR2-p75 Knockdown Lewis Lung Carcinoma Tumor Implants. PLoS ONE, 9(3), e92373.

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Ischemic Muscle Morphology and Angiogenesis

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All mice were euthanized at post-operative day 14. Ischemic and non-ischemic gastrocnemius muscles were gathered and weighed, fixed in 4% paraformaldehyde. Muscle sections (5-μm) were dyed with hematoxylin and eosin (H&E) to examine myocyte morphology. For immunofluorescence assay, tissue sections were stained using anti-CD31 antibody (BD Biosciences, Franklin Lakes, NJ, United States). 10 randomly selected fields from five independent sections in each animal (×400 magnification) were counted, and the vessel density was expressed as number of capillaries/field. To determine local oxidative stress levels, anti-nitrotyrosine antibody (Upstate, Lake Placid, NY, United States) was used. Intensities of fluorescence were measured and analyzed using Image Pro Plus software.

Qi J., Wang J.J., Duan J.L., Lu Z.Y, & Yuan Y.G. (2017). Leonurine Improves Age-Dependent Impaired Angiogenesis: Possible Involvement of Mitochondrial Function and HIF-1α Dependent VEGF Activation. Frontiers in Pharmacology, 8, 284.

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Tumor Tissue Histological Analysis

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Tumor tissues were harvested at the end of experiment and tissues were fixed in 4% paraformaldehyde (PFA) overnight, embedded in paraffin, sliced into 5 μm sections and stained with hematoxylin and eosin (H&E) for histological examination. Tumor tissues were OTC- embedded and sliced into 8μm sections. Frozen tumor sections were fixed in 4% PFA 30-60min, washed with PBS and incubated with 0.5%TritonX-100 to eliminate endogenous peroxidase activity. Then cryosections were blocked with the BSA (5%) and were stained with an anti-CD31 antibody (BD, USA) diluted to 1:400. The TUNEL assays were performed according to the manufacturer’s instructions (Beyotime, china) [17 (link),18 (link)].

Hao J., Xie W., Li H, & Li R. (2018). Prostate Cancer-Specific of DD3-driven Oncolytic Virus-harboring mK5 Gene. Open Medicine, 14, 1-9.

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Immunofluorescence Labeling of CD31 in GC Muscle

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GC muscles were fixed in 2% paraformaldehyde for 1 h at 4 °C and washed three times for 10 min each with PBST (0.2% Triton X-100 in PBS). A small bundle of muscle was carefully dissected by fine forceps from the center portion of the GC muscle. The muscle tissues were then blocked with 1% BSA/PBST (5% normal goat serum) for 1 h at room temperature. The tissues were incubated with rocking overnight at 4 °C with anti-CD31 antibody (1:100 dilution, BD Biosciences, 550274). Next day, tissues were washed overnight in PBS at 4 °C, followed by anti-rat IgG-Alexa 647 conjugate (1:500) at 4 °C for overnight. Immunofluorescence images were acquired using Nikon A1R laser scanning confocal microscope or Nikon 90i fluorescence microscopy.

Li F., Sawada J, & Komatsu M. (2017). R-Ras-Akt axis induces endothelial lumenogenesis and regulates the patency of regenerating vasculature. Nature Communications, 8, 1720.

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Histological and Immunohistochemical Analyses

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Histological analysis was performed using the standard procedure.
For cryosections, dissected tissues or embryos were fixed for 30 minutes in 4% PFA followed by dehydration in 30% sucrose solution for two hours, and then embedded in OCT (Richard-Allan Scientific) and frozen in liquid nitrogen-cooled isopentane. Frozen sections were collected at 7 µm.
For immuonhistochemical and Immunofluorescent analyses, cryosections were stained with antibodies against the following proteins respectively: Mst1, Mst2 (Abcam 51134 and 52641), Ki67 (Novocastra Laboratories NCL-Ki67p) and CD31 (BD 558736) following the standard protocols [38] .
Whole mount staining with anti-CD31 antibody (BD 550274) was performed as described by Takahashi, et al. [39] (link) except for that signals were visualized in a DAB-color developing solution without NiCl_2. Horseradish peroxidase (HRP)-conjugated mouse anti-rat IgG second antibodies were from Santa Cruz.

Du X., Dong Y., Shi H., Li J., Kong S., Shi D., Sun L.V., Xu T., Deng K, & Tao W. (2014). Mst1 and Mst2 Are Essential Regulators of Trophoblast Differentiation and Placenta Morphogenesis. PLoS ONE, 9(3), e90701.

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Immunohistochemical Analysis of Tumor Samples

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FaDu tumors were excised and paraffin-embedded, followed by the standard dehydration process.^{41 (link)} The tumor samples were cut into 5-µm sections and stained with hematoxylin and eosin (H&E). The sections were dewaxed and antigen retrieval was performed in a sodium citrate buffer. The following antibodies were used: anti-FAP (Abcam) and anti-A27 (GenScript Corporation). Biotinylated secondary antibodies (goat anti-rabbit; Jackson ImmunoResearch Laboratories, Suffolk, UK) were used and detection was performed with Vectorstain Elite ABC reagent and Vector ImmPact DAB Peroxidase substrate (Vector Laboratories, Burlingame, CA).
DU145 tumors were excised at 36 dpi, followed by paraformaldehyde fixation, and then cut into 100-µm sections. The blood vessels and cell proliferation were detected with anti-CD31 antibody (BD Pharmingen, San Jose, CA) and anti-Ki67 antibody (BD Pharmingen), respectively.
The examination of tumor sections was conducted with an MZ16 FA fluorescence stereomicroscope (Leica, Buffalo Grove, IL) equipped with a digital charge-coupled device camera (Leica). Digital images (1,300 to 1,030-pixel images) were processed using Adobe Photoshop 7.0 software (San Jose, CA).

Huang T., Wang H., Chen N.G., Frentzen A., Minev B, & Szalay A.A. (2015). Expression of anti-VEGF antibody together with anti-EGFR or anti-FAP enhances tumor regression as a result of vaccinia virotherapy. Molecular Therapy Oncolytics, 2, 15003-.

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