Plasminogen

The uterine samples were homogenized in a lysis buffer (Kurabo, Osaka, Japan). Then, the homogenates were centrifuged, and the resultant supernatants were collected. Western blotting was performed as described previously 3 (link). Briefly, membranes were incubated at room temperature for 1 h with primary antibodies against plasminogen (1 : 1,000; GeneTex, Hsinchu City, Taiwan), plasmin (1: 1,000; GeneTex), tPA (1:1000; Abcam), matrix metalloproteinase (MMP)-12 (1: 1,000; Proteintech Rosemont, IL, USA), angiostatin (1: 1,000; Abcam), or β-actin as a loading control (1 : 5,000; Sigma-Aldrich, St. Louis, MO, USA). The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were acquired using Multi Gauge software (Fujifilm, Greenwood, SC, USA).

Valvular staining was performed on randomly selected 50 AS and 5 control valves. Lipid detection was performed using Sudan black dye followed by counterstaining with nuclear fast red solution. Immunofluorescence was conducted according to the previously described protocol [16 (link)] using primary antibodies against PAI-1 (1:500; Abcam, Cambridge, UK), tPA (1:100; Novus Biologicals, Centennial, CO, USA), α₂-antiplasmin (1:250; Santa Cruz Biotechnology, Dallas, TX, USA), plasminogen (1:500; GeneTex, Irvine, CA, USA), fibrin degradation products (D-dimer, 1:100; Bioss Antibodies, Woburn, MA, USA) and NF-κB (p65, 1:500, Abcam) and the corresponding secondary donkey or goat antibodies conjugated with AlexaFluor 488 or 594 (Abcam) (1:1000). Double-label immunofluorescence analysis was performed for PAI-1 and NF-κB. A negative IgG isotype control was performed routinely. The percentage of immunopositive areas and the fluorescence intensity (FI) were calculated as described previously [16 (link)]. The data were analyzed by two independent investigators blinded to the sample origin. The intra- and inter-observer variability was below 7%.