Anti-VEGF Receptor-3 Antibody, extracellular domain, clone 9D9F9

175 kDa

Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. {Bando, 2004}. Jussila et al reported that VEGFR3 antibodies specifically stain endothelial cells of lymphatic vessels and vessels around tumonrs such as lymphoma and in situ breast carcinoma. {Jussila, 1998}.Interestingly, the spindle cells of several cutaneous nodular AIDS-associated Kaposi′s sarcomas and the endothelium around the nodules were also VEGFR-3 positive. The first specific molecular marker for the lymphatic endothelium should provide a useful tool for the analysis of lymphatic vessels in malignant tumors and their metastases and the cellular origin and differentiation of Kaposi′s sarcomas. {Jussila, 1998}.

Recombinantly produced, purified, hVEGFR-3 extracellular domain protein from baculovirus culture {Jussila, 1998}.

Anti-VEGF Receptor-3 Antibody, extracellular domain, clone 9D9F9 is an antibody against VEGF Receptor-3 for use in FC, IF, IH(P), IP & WB.

Immunohistochemistry: frozen cryosections, 5μm cryosections were air-dried and fixed in cold acetone for 10 minutes. Blocked with 5% normal horse serum and then incubated with 9D9 for 2 hours at room temperature in a humid chamber. Typical dilutions will range from 1:100-1:1000 depending upon lot titer. Detection is with enhanced methods (ABC or poly HRP) and enzymatic substrates for best results.

Immunohistochemistry in paraffin: 5μm thick lightly fixed formalin tissues were processed through decreasing ethanol concentrations, washed with water, then heated in a microwave in 10mM sodium citrate pH 6.0 at 780W for 5 minutes, followed by 450W for 10 minutes. The sections were then incubated in methanol containing 30% hydrogen peroxide for 30 minutes and processed further like the cryosections. {Jussila, 1998}.

Western Blot: antibody recognizes VEGFR-3 on reduced samples of HUVEC and HDMEC cells {primay human umbilical vein endothelial cells & primary human dermal microvascular endothelial cells}. The bands seen at molecular weight 195kDa and 125kDa represent the uncleaved and proteolytically cleaved forms of VEGFR3. The band at 175kDa is considered to be intracellular, unglycosylated precursor {Bando, 2004}.

Immunoprecipitation: 1-5μL per 300-500μL of cell lysate. Use protein A or rabbit anti-mouse IgG capture for best results.

ELISA: 9D9F9 has been used as the detection antibody for a VEGFR-3 ELISA {Bando, 2004}.

Flow Cytometry: reactive on transfected NIH-3T3 cells expressing hVEGFR3, Other cell lines untested. {Jussila, 1998}.

Optimal dilutions will need to be determined by the enduser.

Specific for human VEGFR-3. Western blot and ELISA demonstrate that the antibody is highly specific for VEGFR-3 with no cross-reactivity with human VEGFR-1 or VEGFR-2 protein {Bando, 2004}. Cross reactivity with other species VEGFR-3 proteins untested.

UnPurified ascites fluid containing 0.1% sodium azide as a preservative.

Control
Human lymphatic tissues

VEGFR-3 is not expressed in HEVs of the lymph nodes. {Jussila, 1998]. In lymphomas staining is seen mostly in collapsed vessels in the cortex of lymph nodes infiltrated by the lymphoma cells. The antibody specifically detects lymphatic endothelium in human lymph node and other organs such as tonsil. See Jussila et al, 1998 for complete tissue characterization.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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