Celltracker fluorescent dye

Immunohistology was performed as described previously (Quan et al., 2010). Briefly, cryo‐sections (7 μm) were fixed in 5% paraformaldehyde for two hours at room temperature and were incubated with 0.5% Nonidet P‐40 and then blocked with 2% bovine serum albumin (BSA). The slides were washed with PBS five times and incubated with Col‐1 primary antibody (Santa Cruz Biotechnology) for 1 h at room temperature, followed by incubation with Super Sensitive MultiLink (BioGenex, Fremont CA, USA) for 10 min and SS Label (BioGenex) for 10 min. Then, the slides were developed with One Step AEC Soln (BioGenex) for 3 min and counterstained with hematoxylin (BioCare, Concord, CA, USA) for 20 s and mounted with Supermount (BioGenex). Control staining was performed with corresponding rabbit immunoglobulin and confirmed no immunoreactivity (data not shown). Cells morphology was assessed by incubation of cultures with CellTracker fluorescent dye (Molecular Probes, Eugene, OR, USA) for one hour. The cells were washed with PBS and were fixed in 2% paraformaldehyde for 30 min. Fibroblasts were imaged by fluorescence microscopy. For Phalloidin (Sigma) staining, cells were washed with PBS and were fixed in 2% paraformaldehyde for 30 min followed by Phalloidin staining for 1 h.

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.