Anti fade fluorescent mounting medium

Cells were seeded on Millicell EZ 4-well glass slides (Millipore, MA, USA). After treatment for indicated time, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Cells were then incubated with primary antibodies at 4°C overnight, and secondary antibody for 1 hour at room temperature. The sources and dilutions of antibodies are: mouse anti-α-SMA (1:50, Abcam), rabbit anti-fibronectin (1:200, Abcam), rabbit anti-Vimentin (1:100, Cell Signaling Technology), Alexa Fluor 555-conjugated anti-mouse IgG (1:1000, Cell Signaling Technology), Alexa Fluor 555-conjugated anti-rabbit IgG (1:1000, Cell Signaling Technology), and Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000, Cell Signaling Technology). Cell nuclei were stained with 50 ng/ml 4’,6-diamidino-2-phenylindole (DAPI) for 5 min. Slides were mounted with anti-fade fluorescent mounting medium (Applygen, #C1210). Images were acquired by a Zeiss LSM 510 confocal laser scanning microscope (CLSM, Carl Zeiss, Germany) and processed by Adobe Photoshop CS6.