Rabbit anti human vwf antibody

HUVECs pretreated with DMSO, scrambled and PP2B peptides as described above were then challenged with histamine (5 or 10 μM) or thrombin (0.05 or 0.5 U/ml) for an additional 30 minutes. Cells without any agonist treatment served as a control.
In some studies, cells were treated with 5 mM H202, which blocks VWF secretion by inhibiting N–ethylmaleimide sensitive factor (NSF) [18 (link)]. In some experiments, scrambled and PP2B peptides were added 30 minutes after histamine treatment and incubated for an additional hour. The VWF antigen level in cell supernatants was analyzed using a standard ELISA technique. Samples were incubated for 2 hours on microtiter plates pre-coated with 1 μg/ml rabbit anti-human VWF antibody (Dako, CA). After several washes, samples were incubated with 2 μg/ml of HRP conjugated anti-human VWF antibody (Dako, CA) and color-developed with the addition of TMB (3, 3′, 5, 5′-tetramethylbenzidine; Thermo Fisher Scientific, MA). The reaction was stopped with 1 M HCl and the absorbance was read at 450 nm. Absorbance was converted to % of plasma VWF by comparing the absorbance values to a standard curve generated from human plasma samples. Fold differences in the VWF antigen levels were determined after normalizing the VWF values obtained for test samples (peptide and/or agonist treatment) to that obtained from media wherein cells were left untreated.

Plasma vWF antigen was determined by latex immunoassay (LIA). Plasma vWF activity was determined by collagenbinding assay using rabbit-anti-human vWF antibody (DAKO, NY, USA) for detection and type III collagen from placenta (Thermo, NY, USA) for capturing.