In situ TF staining of representative human thrombi.

Human arterial or venous thrombi were fixed overnight in 4% paraformaldehyde immediately after thrombectomy and were paraffin-embedded. Five-micrometer sections were deparaffinized and rehydrated, and TF was stained by using digoxigenin-labeled FVIIa (100 nM, 2 h, 37°C) in Tris-buffered saline (TBS) containing 5 mM Ca⁺⁺, followed by incubation with sheep Fab antidigoxigenin antibody conjugated with horseradish peroxidase (1:1000 dilution, 1 h, 37°C). Color was developed using diaminobenzidine tetrahydrochloride (DAB) (A-D). Immunohistochemical staining was performed on 5-μm sections blocked with 1% peroxyde in methanol and appropriate normal serum and incubated with rabbit polyclonal antihuman TF antibody (1 μg/mL, 2 h, 37°C), stained by a biotin streptavidin–amplified detection system, developed with DAB, and counterstained with hematoxylin (E-G). Another series of nonfixed human arterial or venous thrombi were maintained immediately after thrombectomy in 15% sucrose overnight at 4°C. Then cryosections (8 μm) were blocked with bovine serum albumin (BSA) and incubated with HTF1-7B8, a murine monoclonal anti-TF antibody (20 μg/mL, 1 h, room temperature) followed by a gold-labeled goat antimouse IgG and silver enhancement of the colloidal gold. The sections were embedded in fluorescent mounting medium (I). An irrelevant IgG rabbit mAb was used as a control at equivalent concentration of the specific antibodies (H,J).