Sheep anti vwf

For detection of vWF, 2µl of plasma was electrophoresed and probed with sheep anti-vWF 1:1000; Abcam, MA, USA). Western blotting was carried out as published (26 (link)). For VCAM-1, a total of 40µg of protein from liver and gut lysates was resolved in 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad Laboratories Pty Ltd, Australia) and rabbit anti-VCAM-1 (1:1000; Abcam, USA) and rabbit anti-eNOS (1:500; Abcam, USA) was used. For plasma von Willebrand Factor (vWF) analyses, 2µl of sample were resolved in 7.5% polyacrylamide gels, and sheep anti-vWF 1:1000; Abcam, MA, USA) was used to detect vWF. A standard curve was created using known concentrations of recombinant vWF assessed via western blot, and the amount of vWF was calculated from that curve.

The caspase-1 activity in the carotid arterial endothelium was analyzed in situ by labeling the active caspase-1 proteins with Fluorescent Labeled Inhibitor of Caspases (FLICA™) probes (ImmunoChemistry Technologies, LLC, Bloomington, MN) as described previously (34 (link)). The FLICA probes are comprised of three moieties including a caspase-1 recognition sequence tyrosine-valine-alanine-aspartic acid (YVAD) that binds to active caspase-1, a fluoromethyl ketone moiety that results in irreversible binding with the enzyme, and a fluorescent tag carboxyfluorescein reporter. After entering the cells, the FLICA reagent carboxyfluorescein-YVAD-fluoromethyl ketone becomes covalently coupled to the active caspase-1, whereas any unbound FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the active caspase-1 enzyme activity in the cell or tissue samples. To detect caspase-1 activity in the carotid arterial endothelium, frozen artery section slides were first fixed in acetone and incubated overnight at 4°C with sheep anti-vWF (1:200 dilution; Abcam, Waltham, MA). These slides were then costained with fluorescence-conjugated anti-sheep secondary antibody and FLICA reagent (1:10 dilution) for 1.5 h at room temperature, washed, mounted, visualized, and analyzed by confocal microscopy as described previously.