Immunofluorescence and Microscopy Analysis of Cell Migration and ECM Remodeling

Immunofluorescence was performed as described previously [19] (link). Phospho-MLC2 (Ser 19) antibody was used at 1∶50 dilution. FSP1/S100A4 antibody (Millipore) was used at 1∶200 dilution. Samples were observed with an Olympus IX81-ZDC-DSU microscope equipped with a cooled CCD camera (ORCA-ER, Hamamatsu), and the imaging system was driven by MetaMorph software (Universal Imaging). For Time-lapse microscopy, 44As3 cells (1×10⁴) and CaF37 cells (2.5×10⁴) were plated onto 3D Matrigel (120 µl/well) solidified in a multi-well glass bottom dish (Matsunami). To quantitate ECM remodeling, digital images were converted using ImageJ 1.41o and the movement of microbeads was analyzed. Total distance moved during the time-lapse imaging was calculated for each microbead and normalized by the average value in 44As3 cells. The total and net migration distances of cells before the formation of invasive foci were determined from the time-lapse movies with the manual tracking function of the ImageJ 1.41o software.