Anti von willebrand factor

Liver samples were fixed in 10% formalin, embedded in paraffin and sectioned. After antigen retrieval procedure and endogenous peroxidase activity inhibition, sections were incubated with anti-von Willebrand Factor (1:400; Dako, Glostrup, Denmark) or anti-P-selectin (1:400; Biovision, Milpitas, CA) 1 h at room temperature. HRP-Rabbit/Mouse (Dako) secondary antibody was added. Colour development was induced by incubation with a DAB kit (Dako) and the sections were counterstained with hematoxylin. Sections were dehydrated and mounted. The specific staining was visualized and images were acquired using a microscope equipped with a digital camera and the assistance of Axiovision software. vWF relative volume was determined by point-counting morphometry on immunoperoxidase-stained sections, using a point grid to obtain the number of intercepts over vWF positive cells over the tissue. Six fields were counted in each liver. All measurements were performed by two independent blinded observers. The relative volume was calculated by dividing the number of points positive in sinusoidal areas by the total number of points over liver tissue.

Tissues were formalin fixed and paraffin embedded. Paraffin sections were first deparaffinized, and then steamed for 40 minutes in citrate buffer, pH 7, at 95°C. Sections were immunostained using Dako ARK (Animal Research Kit) (Dako, Glostrup, Denmark). Diaminobenzidine reaction was used for visualization, followed by haematoxylin counterstain. Primary antibodies used were: anti-von Willebrand factor (DAKO, Glostrup, Denmark), anti-MYCN (Novus Biologycals, Littleton, USA). To quantify protein expression three high-power (x400) fields of the highest stained area were digitally captured and pixel values quantified using Image J software.

To determine IEL and IT formation, paraffin tissue sections were stained with elastica van Gieson, as recommended by the manufacturer (Muto Pure Chemicals, Tokyo, Japan). Immunohistochemical analysis was performed as described previously [8 (link), 9 (link)]. Rabbit polyclonal anti-t-PA (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-von Willebrand factor (Dako Cytomation, Santa Clara, CA, USA), mouse monoclonal anti-α-SMA (Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-MMP2 (Novus Biologicals, Little, CO, USA) antibodies were used.