Immunofluorescence Staining
CFs were cultured on sterile glass coverslips were fixed in 4%
paraformaldehyde for 10 min, washed with PBS three times, and
permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, Saint Louis, MO)
in PBS for 20 min. After blocking with 10% goat serum (Jackson
ImmunoResearch, West Grove, PA) at room temperature for 30 min. Then,
the cells were incubated with rabbit anti-vimentin (1:200), mouse
anti-α-SMA (1:400) overnight in humidified incubator. The slides were
then washed with PBS and incubated with Alexa Fluor 594 conjuncted goat
anti-mouse IgG (1:400) or Alexa Fluor 488 conjuncted goat anti-rabbit
(1:200; Jackson, West Grove, PA) for 30 min at 37 oC.
The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI,
1:1000; Sigma-Aldrich, Saint Louis, MO). Finally, the slides were
mounted in fluoromount-G(Southern Biotech), and examined using a
fluorescence microscope.