Tmb 3 3 5 5 tetramethylbenzidine

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.

Serum pool of five animals was used for antibody detection. For IgM and global IgG analysis, goat anti-mouse IgM or IgG (SouthernBiotech, AL), 1 μg/mL diluted in PBS, were pre-immobilized to half-area-high binding 96 well ELISA plates, following incubation at 4 °C overnight. Afterwards, the plates were subjected to four washes with PBS and blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour and 30 minutes at room temperature. BSA was removed and the plates were washed with PBS, serum samples were diluted 1:100 in PBS-1% BSA and serially three-fold and five-fold diluted to determine the concentration of IgM and IgG, respectively. IgM and IgG (1 μg/mL) diluted 1: 3 and 1: 5, respectively, were used as the standard curve. The samples were incubated at 4 °C overnight. Plates were washed four times with PBS and incubated with 50 μL of goat anti-mouse IgM (1:4000) and IgG (1:8000) (SouthernBiotech, AL) conjugated to peroxidase (HRP) diluted in PBS 1% BSA and incubated at room temperature for 1 h 30 min. At the end, the plates were again subjected to four washes with PBS and then developed with TMB (3,3′, 5,5;-tetramethylbenzidine) (Thermo Fisher); the reaction was stopped with 50 μL of 3 N HC1 and the reading performed on a 450 and 650 nm filter microplate reader (iMark, BioRad, US).