Live dead double staining solution

To detect the remains of the cells, samples of dECM-SF and multi-channeled dECM-SF (MC dECM-SF) hydrogel were examined under fluorescence microscopy after DAPI (4′,6-diamidino-2-phenylindole) staining. For cell viability, the scaffolds were incubated with live/dead double staining solution (Sigma, USA) for 30 min and then observed by confocal microscopy (Leica, Heidelberg, Germany).

The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned into 5 µm sections. The sections were stained with hematoxylin and eosin (H&E) to evaluate their structures and stained with Safranin O-Fast Green to visualize glycosaminoglycan (GAG) deposits. The expression of type II collagen was detected by mouse anti-human type II collagen monoclonal antibody (1:200; Abeam, Cambridge, MA, USA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:50, Dako, Denmark) and color development with diaminobenzidine tetrahydrochloride (DAB, Dako, Denmark). To detect the existence of DNA, samples of the dECM-SF scaffold and MC dECM-SF scaffold were examined under fluorescence microscopy after DAPI (4′,6-diamidino-2-phenylindole) staining. For cell viability, the scaffolds were incubated with live/dead double staining solution (Sigma, USA) for 30 min and then observed by confocal microscopy (Leica, Heidelberg, Germany).