Vwf antibody

Cadmium granules, potassium nitrate, bovine thrombin, sodium chloride, calcium chloride, lipopolysaccharide (LPS, from Escherichia coli serotype 026: B6), trichloroacetic acid, trizma base, sulphorhodamine B, 3′diaminobenzidine, HEPES, and other reagents were purchased from Sigma Aldrich, USA. Avidin peroxidase and streptAvidin peroxidase, anti-VCAM-1, anti-ICAM-1, anti-E-Selectin antibodies were obtained from Santa Cruz Biotechnology, USA. 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Scyteck, USA. Chromogenic substrates from Chromogenix AB, Italy. Plasmin, uPA and double chain tPA standards (tcu-PA) were obtained from Sekisui, USA. Mouse IL-6 minikit and mouse TNF-α minikit, recombinant mouse TNF-α were purchased from Thermo Scientific, USA. Fetal bovine serum from Gibco, BRL, USA. vWF antibody from DAKO, USA. Murine TF standard were obtained from R&D Systems, USA.

1×10⁶ 143B cells were mixed with 500 μg 10H10, 5G9 (mouse monoclonal antibodies against TF) or a control mouse IgG in PBS and injected subcutaneously (n=6) in NOD-SCID mice (Jackson Laboratories, Westgrove, PA, USA). Tumor growth was measured over time using calipers; tumor volume was calculated as width² * length * 0.5. Tumors were harvested after 3 weeks and formalin-fixed, paraffin-embedded for further analysis. Lungs were snap-frozen in liquid nitrogen. Animal experiments were approved by the animal ethical committee of the Leiden University Medical Center. Immunohistochemsirty was performed as described above, a vWF antibody (1:5000, DAKO, Glostrup, Denmark) was used to stain microvessels. Ki67 (1:800, DAKO, Glostrup, Denmark) was used to proliferating cells. Ki67 positive nuclei were quantified as described by Tuominen et al.(23 (link)) Microvessel density was quantified by counting vWF+ microvessels in at least two fields per tumor at 10× magnification. Means were calculated, a t-test was performed to determine statistical significance between IgG control and 10H10 or 5G9 groups.