Cells were fixed with ice-cold methanol for 15 min. Cells were then washed and blocked in 5% normal donkey Serum (Jackson Research Laboratories, Inc., West Grove, PA) for 1 h at room temperature, followed by mouse monoclonal αSMA antibody (1:200 dilution, Dako, Carpinteria, CA) for 90 min. Cells were rinsed and then incubated in secondary antibodies Alexa Flour ® 488 donkey anti-mouse (Invitrogen, Eugene, OR) for 1 h at room temperature. The cells were washed three times with PBS, mounted in Vectashield containing 4′−6-diamidino-2-phenylindole (DAPI; Vector Laboratories), and photographed with a Leica DM 4000B fluorescent microscope (Leica, place, state, country) equipped with a digital camera (SpotCam RT KE).
For Smad2/3 staining, HCF cells (1.8 × 103 in 300 μl medium/well) were grown to sub-confluency in each well of 4-well Nunc Lab-Tec chamber slides (Nunc, Rochester, NY). Cells were treated with or without TRAM34 (25 μM) for 24 h. The cultures were washed and treated with TGFβ−/+ (5 ng/ml) for 1 h. Cells were fixed with ice-cold methanol for 15 min and immunohistochemistry for smad2/3 protein was performed using mouse monoclonal anti-smad2/3 antibody (1:100 dilution in PBS; Santa Cruz Biotechnology, Santa Cruz, CA) followed by the incubation with secondary antibody Alexa Flour® 488 donkey anti-mouse (Invitrogen, Eugene, OR) was used, respectively.
For Smad2/3 staining, HCF cells (1.8 × 103 in 300 μl medium/well) were grown to sub-confluency in each well of 4-well Nunc Lab-Tec chamber slides (Nunc, Rochester, NY). Cells were treated with or without TRAM34 (25 μM) for 24 h. The cultures were washed and treated with TGFβ−/+ (5 ng/ml) for 1 h. Cells were fixed with ice-cold methanol for 15 min and immunohistochemistry for smad2/3 protein was performed using mouse monoclonal anti-smad2/3 antibody (1:100 dilution in PBS; Santa Cruz Biotechnology, Santa Cruz, CA) followed by the incubation with secondary antibody Alexa Flour® 488 donkey anti-mouse (Invitrogen, Eugene, OR) was used, respectively.