Mouse anti human vimentin

The wound-healing assay was performed as previously described with minor modifications (20, 21) . Briefly, cells were grown to 100% confluency in P60 culture dishes, and starved in serum-free culture medium for 24 h before scratching. A circle the cell layer of the monolayer approximately 500 µm in diameter was then made by scrapping with a 10-µl extra-long micro-pipette tip. The cells were washed twice with PBS and incubated with serum-free culture medium with or without TGF-β1 and metformin. Microphotographs were acquired with a digital camera at 0, 12 and 24 h after scratching, and the cell-free area was measured using ImageJ software (version 1.51). The migration area was evaluated as the cell-free area at 12 and 24 h, as a percentage of the scratch at 0 h. Immunocytochemistry. After the PANC-1, MIAPaCA-2, and BxPC-3 cells were grown to >80% confluency in 35-mm µ-dishes (Ibidi), they were washed with PBS and fixed with 4% paraformaldehyde for 20 min at 25˚C. The cells were then incubated with mouse anti-human-vimentin (Santa Cruz Biotechnology, Inc.) for 2 h at 25˚C followed by 1 h of incubation with anti-rabbit IgG conjugated with Alexa Fluor 594 (Alexa Fluor 594, Life Technologies; Thermo Fisher Scientific, Inc.) at 25˚C. The cells were observed using an epi-illumination on a laser scanning confocal microscope (Olympus Corp.).