Vector blocking kit

4-μm paraffin sections were deparaffinized in xylol and rehydrated in graded alcohol series. Endogenous peroxidase was inhibited using 3% H₂O₂ in methanol. The sections were then washed in distilled water and heated in a microwave oven (in citrate buffer 10 mM, pH 6, for CD31, and EDTA 1 mM, pH 7.5, for factor VIII) 15 min for epitope retrieval. No pretreatment was needed for D2-40. Slides were incubated first with normal horse serum (1/30 avidin 10%) for CD31 and with normal goat serum (10% avidin) for factor VIII and Fli-1 for 5 min and then in biotin for 10 min. Endogenous biotin was inhibited with a Vector Blocking kit (Vector Laboratories; Burlingame, CA). The slides were then incubated at 20C for 40 min with monoclonal antibodies for CD31, the slides were incubated overnight at 8C. The slides were incubated with anti-mouse/rabbit biotinylated bridging antibodies (dilution 1/200) for 30 min. Sections were then washed and incubated with standard avidin-biotin complex (ABC; DakoCytomation, Glostrup, Denmark) for 30 min. Antibody binding was revealed using H₂O₂ as a substrate and diaminobenzidine as chromogen. Counterstaining was performed with hematoxylin.