F6140

Uninjured mice were sacrificed at the age of 10 weeks. In DMM or sham surgery groups, animals were sacrificed at either 2 weeks or 8 weeks postoperatively. Specimens were fixed in 4% paraformaldehyde for 24 hours, decalcified with 14% ethylenediaminetetraacetic acid (EDTA) for 14 days, and embedded in the optimum cutting temperature (OCT) compound. Sections were prepared at 6 μm thickness with a Cryofilm type 3c (SECTION-LAB, Japan).
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for CD31 (1:50, ab28364, Abcam, MA, USA), α-SMA (1:400, ab7817, Abcam), ED-A fibronectin (1:400, F6140, Sigma Aldrich, MO, USA) or TGF-β1(1:400, ab92486, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083 or ab150119, Abcam), and mounted with mounting medium containing DAPI (H-1500, Vector Labs). Bright field images were obtained on a Leica DM 6B microscope (Leica Biosystems, Germany). Immunofluorescent images were acquired on a LSM 780 FCS confocal microscope (Carl Zeiss, Germany).

Plasma samples from the patients with VTE were obtained from Dr. Anetta Undas (Institute of Cardiology, Jagiellonian University, Kraków, Poland). Cellular Fn-EDA levels in the plasma were measured by sandwich enzyme-linked immunosorbent assay (ELISA) as described.[14 (link)–16 (link)] Briefly, microtiter plates were coated overnight at 4°C with primary antibody for Fn-EDA (3E2, 10 μg/mL, Sigma, catalog # F6140) diluted in 50 mM sodium carbonate buffer. 10 μl of plasma samples (diluted 1:1 in PBS) were incubated for 2 h in the coated wells at 37°C. After five washes, biotinylated secondary antibody to Fn (2 μg/ml diluted in blocking buffer, Sigma, catalog # F3648) was added to wells and incubated for 1 hour at room temperature. The avidin HRP solution (1:1000) in the blocking buffer was added to wells and incubated for 30 minutes following five washes. Micro titer plates were washed five times before adding 3, 3’, 5, 5’-tetramethylbenzidine substrate solution (Sigma, catalog # T0440) to the wells, and the colorimetric reaction was stopped with 2 M H₂SO4 after 10 min. Results were read in an ELISA microplate reader at A450 nm. Human cellular Fn (Sigma, catalog # F2518) was used for standards.