Sp8 confocal microscope with 2 photon

Cell morphology was assessed by incubation of cultures with CellTracker® fluorescent dye (Molecular Probes, Eugene, OR, USA) for 1 h. The cells were washed with PBS and were fixed in 2% paraformaldehyde for 30 min. For Phalloidin staining, cells were washed with PBS and were fixed in 2% paraformaldehyde for 30 min followed by Phalloidin stain (Sigma) for 1 h. For immunohistology, cryosections (7 μm thickness) were fixed in 2% paraformaldehyde for 2 h at room temperature and were incubated with 0.5% Nonidet P-40, then blocked with 2% bovine serum albumin (BSA). The slides were washed with PBS five times and incubated with MMP-1 and HSP47 primary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Cells were washed and then incubated with secondary antibody for 30 min at room temperature. Images were obtained using Zeiss fluorescence microscopy. Second harmonic generation microscopy was performed using a Leica SP8 Confocal Microscope with 2-Photon, at University of Michigan Microscopy and Image Analysis Laboratory.