2.7. Immunofluorescence Staining of ECM Protein Deposition

On day 9, the hydrogels containing NHDFs were washed three times in DPBS and fixed in 3.7% paraformaldehyde (PFA) in DPBS for 30 min. Subsequently, the hydrogels were immersed in 0.1% Triton X-100 in DPBS for 30 min at room temperature to permeabilize the cell membranes. For collagen type I and elastin staining, the hydrogels were blocked in 10% horse serum in DPBS for 1 h. The monoclonal mouse anti-collagen type I antibody (Abcam, Cambridge, UK) at 1/500 dilution in 10% horse serum was added to the hydrogels and the samples were incubated at 4 °C for 12 h. The hydrogels underwent 3×10 min washes in DPBS before incubation in 1/200 dilution of Alexa Fluor 555 Goat Anti-Mouse IgG Secondary Antibody (Abcam, Cambridge, UK) in 10% horse serum in DPBS for 3 h at room temperature. A similar procedure was used to stain elastin, using a primary monoclonal rabbit anti-human elastin antibody (Abcam, Cambridge, UK) and Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (Abcam, Cambridge, UK). For F-actin cytoskeleton staining, after 3 washes in DPBS and hydrogels were soaked in a solution with Alexa Fluor 568 phalloidin at 5 IU/mL (Invitrogen, Cambridge, USA) for 90 min incubation at room temperature. Cellular nuclei were stained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Califonia, USA). Confocal imaging was performed using a CLSM, Leica SP2 inverted microscope (Wetzlar, Germany). The immunofluorescence staining images of cell-free hydrogels were shown in supporting information.