2.6. Cytoskeleton staining assays
12-well plate cell were coated with
1noml/mL of EGF, RHC and RHC/EGF at 4℃ overnight. After washing with
phosphate-buffered saline (PBS), the wells were blocked with 1% (w/v)
bovine serum albumin for 30 min. NIH/3T3 cells were seeded in the same
plates at 4 ×104 cells per well in FBS-free medium.
After 4h, cells were washed with PBS and fixed for 10 min with 4%
paraformaldehyde solution, and then washed with PBS. Cells were stained
with a phalloidin (1:200, Solarbio, China) for 30 min at 37℃ in the
dark. The cells were washed three times with PBS to remove unbound
phalloidin. Cells were stained with DAPI (1:1000, Beyotime, China) for 5
min at room temperature. The samples were washed three times with PBS to
remove unbound DAPI. Images were collected using the LSM 700 confocal
LASER scanning microscope (ZEISS, German), with excitation wave lengths
of 488 and 561 nm. Analyze the image with image J to calculate the cell
adhesion area
2.7. Preparation and characterization of RHC and RHC/EGFfreeze-dried dressing