*4.6. Fluorescence Staining and Confocal Laser Scanning Microscopy*

Cell-seeded scaffolds were fixed with 4% buffered formaldehyde and permeabilized with 0.2% Triton X-100 (Sigma) in Hank's balanced salt solution (HBS) (Gibco). Autofluorescence was blocked with, 30 min incubation in 3% BSA in HBS. Staining of cytoskeleton was performed with Alexa Fluor 488 phalloidin and staining of the nuclei with 0.3 μM DAPI (4 ,6-diamidino-2-phenylindole dihydrochloride; both Invitrogen) in HBS. Samples were imaged using a confocal laser scanning microscope LSM 510 (Zeiss, Jena, Germany) applying an excitation/emission wavelength of 405/461 nm (diode laser) for DAPI and 488/519 nm (argon laser) for Alexa Fluor 488.
