Alexa fluor 488 or 594

HUVECs were cultivated to confluence on glass slipcovers before their fixation with 4% paraformaldehyde for 10 min. Fluorescent labeling antibodies were applied to monolayer cells after blocking with 3% BSA for 30 min at room temperature. Afterwards, sections were incubated with DAPI (Solarbio, China), a nucleus-specific anti-fluorescence agent.
Tissues embedded in paraffin were sectioned and placed on glass transparencies. Sections were immersed in 3% H₂O₂ and serum albumin to inhibit endogenous peroxidase activity and nonspecific binding, respectively. After that, primary antibodies and secondary antibodies conjugated with Alexa Fluor 488 or 594 (Abcam, USA) were added, and the sections were incubated at 4 °C for 24 h. Anti-fluorescence quenching agent DAPI (Solarbio, China) was used to counterstitch nuclei. Image acquisition was performed using a Zeiss LSM510 Meta (Zeiss, Germany).

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%.