2.3. Immunocytochemistry (ICC) of cardiac fibroblasts

Immunocytochemistry was performed on adult cardiac fibroblasts isolated from sham or ACF rats, or normal fibroblasts subjected to mechanical stretch (24 h, 20% stretch, 1 Hz) or exposed to recombinant human chymase (2.5 μg/ml for 2 h at 37 °C, Sigma-Aldrich #C8118). The cells were fixed in 4% formaldehyde (Tousimis, Rockville, MD) for 20 min at room temperature (RT) and washed 3 times in PBS, and permeabilized with 0.1% Triton-X-100 (Fisher #BP-151) for 15 min at RT. 10% normal serum (in 1% bovine serum/PBS) for 1 h at RT was used for blocking, followed by overnight incubation at4 °C with a chymase monoclonal (Abcam #ab2377; 1:50) and a vimentin polyclonal antibody (Millipore #AB5733; 1:500). Alexa Fluor 488- and 594-conjugated secondary antibodies (1:700, Life Technologies/Invitrogen, OR) with the appropriate host combinations were incubated for 1 h to stain. Nuclei were stained with DAPI (1.5 μg/ml; Vector Laboratories, CA). Image acquisition and analyzing were performed using a Leica DM6000 epifluorescence microscope and SimplePCI software as described previously [16].