Von willebrand factor

The middle part of the gastrocnemius muscle and the distal end of crush nerves were harvested and proteins were extracted. Proteins (50 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to blotting membranes. After blocking with nonfat milk, the membranes were incubated with antibodies: S-100 (1:1000 dilution, Merck Millipore, Burlington, MA, USA), neurofilament (1:1000 dilution, Merck Millipore, Burlington, MA, USA), CD 68 (1:1000 dilution, Bio-rad, Hercules, CA, USA), von Willebrand factor (1:200 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), Isolectin B4 (1:200 dilution, Vector Laboratories, Burlingame, CA, USA), desmin (1:1000 dilution, Abcam, Cambridge, MA,USA), acetylcholine receptor (1:1000 dilution, Merckmillipore, Burlington, MA, USA), GAPDH (1: 2000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody and developed using ECL western blotting reagents. The intensity of protein bands was determined by a computer image analysis system (IS1000) (Alpha Innotech Corporation, San Leandro, CA, USA) [29 (link)].

Heme oxygenase-1 was stimulated as described above, inflammation induced and lungs of mice removed after 24 h (n = 4). The circulatory system of the lungs was flushed and lungs with 4% paraformaldehyde (PFA) for 10 min at 25 cmH₂O inflated. Lungs were removed and fixed in PFA for 24 h. Rabbit polyclonal anti-HO-1 was used as primary antibody (Enzo, Life Sciences GmbH, Lörrach, Germany) to mark the enzyme and the endothelial marker von Willebrand Factor (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was employed and additionally DAPI for nuclear staining. Images were visualized by using a confocal microscope (LSM 510, Meta, Carl Zeiss). Images shown are representatives of four experiments and were analyzed using ImageJ, a public program developed at the National Institutes of Health to officially analyze scientific images.

To visualize the presence of EC, antibodies to CD31 (1:1000) (Abcam, Germany) and von Willebrand factor (1:100) (Santa Cruz, Germany) were selected and stained by immunofluorescence, while smooth muscle actin (1:50) (Abcam, Germany) to visualize SMC. The tensile strength of recellularized veins (n = 3), work and elongation was compared to normal and decellularized veins.