Decellularization and ECM Characterization

To confirm decellularization and assess the presence and distribution of glycosaminoglycans (GAGs) and collagen, DNP and native NP tissue samples were fixed, paraffin-embedded, and sectioned at a thickness of 7 μm, prior to staining with either 4′,6-diamidino-2-phenylindole (DAPI), Toluidine blue (0.1%, Sigma), or Picrosirius red (1.3% picric acid and 0.1% direct red 80 (Sigma)), using previously established methods (Shridhar et al., 2019 (link), 2020 (link)). DAPI staining was visualized using an EVOS FL fluorescence microscope (ThermoFisher Scientific Inc.), and Toluidine blue and Picrosirius red staining were visualized using an EVOS XL Core microscope (ThermoFisher Scientific Inc., Burlington, Canada) and a Nikon Optiphot polarizing microscope (Nikon Instruments Inc., Melville, United States), respectively.