VIC-laden hydrogels were frozen in optimal cutting temperature (OCT) compound, and 10-µm cross-sections were obtained. To detect calcium deposition, sections were stained with 0.02 mg/mL Alizarin Red S solution (Sigma). Von Kossa staining (American MasterTech) was used to visualize calcium-phosphate deposition.
Immunofluorescence staining for α-SMA and runt-related transcription factor 2 (Runx2) was performed using anti-human α-SMA antibody (Clone 1A4, Dako) or an anti-human Runx2 antibody (Abcam), followed by biotin-labeled secondary antibody (Vector Labs) and streptavidin-labeled AlexaFluor 488 for α-SMA and AlexaFluor 594 for Runx2. Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Images were taken with an Eclipse 80i microscope (Nikon) and processed with Elements 3.20 software (Nikon). A custom MATLAB-based script was used to detect DAPI-stained cellular nuclei in representative images of calcified valves from four different human donors. Quantification of immunofluorescent signal surrounding and within each nucleus provided information on α-SMA expression and Runx2 expression and nuclear translocation on a cell-by-cell basis.
Immunofluorescence staining for α-SMA and runt-related transcription factor 2 (Runx2) was performed using anti-human α-SMA antibody (Clone 1A4, Dako) or an anti-human Runx2 antibody (Abcam), followed by biotin-labeled secondary antibody (Vector Labs) and streptavidin-labeled AlexaFluor 488 for α-SMA and AlexaFluor 594 for Runx2. Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Images were taken with an Eclipse 80i microscope (Nikon) and processed with Elements 3.20 software (Nikon). A custom MATLAB-based script was used to detect DAPI-stained cellular nuclei in representative images of calcified valves from four different human donors. Quantification of immunofluorescent signal surrounding and within each nucleus provided information on α-SMA expression and Runx2 expression and nuclear translocation on a cell-by-cell basis.