Liver sections were first deparaffinized and hydrated through a series of washing steps from alcohol to distilled water. They were sequentially stained first with Gill II hematoxylin for 3 min and then Eosin Y for 45 s. The stained slides were washed in 95% alcohol, dehydrated, cleared in xylene, and mounted using Cytoseal.
Tissue sections from the liver were also immunostained for VWF using an automated staining system (Dako Cytomation, Carpinteria, CA, USA). Briefly, antigen was first retrieved by incubating tissue sections in a sodium citrate buffer (10 mM, pH 6.0) containing 0.05% Tween-20 for 20 min. The sections were quenched of endogenous peroxidase activity by 0.3% hydrogen peroxide (15 min) and blocked with a serum-free protein blocking solution for 5 min. They were then incubated with either a rabbit anti-VWF (Agilent Dako, Santa Clara, CA USA), mouse anti-fibrin (Agilent Dako), or control IgG for 60 min at room temperature, followed by extensive washing with Tris buffer saline (pH 7.6). The sections were incubated with Envision anti-rabbit or anti-mouse HRP polymers (Agilent Dako) for 30 min, followed by 8 min incubation with DAB substrate (Agilant Dako). After being counterstained in Hematoxylin, the sections were mounted with Cytoseal.
Tissue sections from the liver were also immunostained for VWF using an automated staining system (Dako Cytomation, Carpinteria, CA, USA). Briefly, antigen was first retrieved by incubating tissue sections in a sodium citrate buffer (10 mM, pH 6.0) containing 0.05% Tween-20 for 20 min. The sections were quenched of endogenous peroxidase activity by 0.3% hydrogen peroxide (15 min) and blocked with a serum-free protein blocking solution for 5 min. They were then incubated with either a rabbit anti-VWF (Agilent Dako, Santa Clara, CA USA), mouse anti-fibrin (Agilent Dako), or control IgG for 60 min at room temperature, followed by extensive washing with Tris buffer saline (pH 7.6). The sections were incubated with Envision anti-rabbit or anti-mouse HRP polymers (Agilent Dako) for 30 min, followed by 8 min incubation with DAB substrate (Agilant Dako). After being counterstained in Hematoxylin, the sections were mounted with Cytoseal.