Millicell ez 24 well glass slides

Cells were seeded on Millicell EZ 24‐well glass slides (Millipore). After treatment for the indicated times, the cells were fixed with 4% paraformaldehyde, permeabilized with methanol/acetone (1:1), and blocked with normal goat serum. Then, the sections were incubated with anti‐fibronectin (1:200, Abcam) and anti‐α‐SMA (1:200, Abclonal) prepared with goat serum. After washing in PBS, the sections were incubated with fluorescein goat anti‐rabbit IgG or fluorescein goat anti‐mouse IgG (1:200, Cell Signaling Technology). Cell nuclei were stained with 50 ng mL^–1 4’,6‐diamidino‐2‐phenylindole (DAPI) for 5 min. Slides were mounted with anti‐fade fluorescent mounting medium (Applygen). Images were acquired by a Zeiss LSM confocal laser scanning microscope (CLSM, Carl Zeiss) and analyzed with ZEN2.1 software.

Cells were seeded on Millicell EZ 24-well glass slides (Millipore). After PBS wash, cells were fixed with 4% paraformaldehyde, permeabilized with methanol/acetone 1:1, and blocked with normal rabbit or goat serum. Then the slides were incubated with the anti-CREB (#MA1-083, Invitrogen Inc.) and anti-p-CREB antibody (#9198, Cell Signaling Technology) or normal rabbit IgG at 4°C overnight. After the PBS washings, the slides were incubated with fluorescence goat anti-rabbit IgG or fluorescence goat anti-mouse IgG (1:200, 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). Images were acquired by a Zeiss 800 confocal microscope (CLSM, Carl Zeiss, Germany) and processed by ZEN software.