Supernuclease

In order to compare the decellularization effects of detergents, the porcine corneal stroma was separately treated with 0.5% SLG (Sigma-Aldrich, St. Louis, MO, USA), SLS (Sigma-Aldrich), SDS (Solarbio, Beijing, China), or Triton X-100 (Solarbio), supplemented with 200 U/mL supernuclease (Sino Biological, Beijing, China) in PBS for 2 h at room temperature under shaking condition (100 r/min).
In order to compare the characterization of DPCs, the native porcine corneal stroma was treated with one of three decellularization methods as described below. Method A was the decellularization method we presented in this study by using SLG and supernuclease. Methods B and C were two traditional decellularization methods reported by previous studies.^{14 (link),15 (link)} A summary of the decellularization methods is shown in Table 1.

Cells were lysed in RIPA lysis buffer with fresh protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and SuperNuclease (SinoBiological, Beijing, China) on ice for 30 min. We measured protein samples using a standard bicinchoninic acid assay (Thermo Fisher, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene fluoride membranes. The membranes were blocked with tris-buffered saline with 0.1% Tween® 20 Detergent plus 5% nonfat dry milk for 2 h, followed by incubation with primary antibodies overnight at 2–8°C. All primary antibodies were purchased from Abcam (Cambridge, UK) or Cell Signaling Technology (Danvers, MA, USA) unless otherwise indicated. Species-specific horseradish peroxidase-conjugated secondary antibodies (1:10,000) were incubated with the membranes for 1 h at room temperature. Immunoreactive signals were detected using an enhanced chemiluminescence (ECL) system using an ECL kit (Millipore, Burlington, MA, USA). All loading samples were normalized to the internal reference protein, glyceraldehyde 3-phosphate dehydrogenase.