Sm22α

After methanol/acetone fixation, fibroblasts were washed with PBS solution and incubated with primary antibodies (αSMA: mouse monoclonal IgG2a, #M0851; Dako, Denmark; SM22α: polyclonal rabbit IgG, ab14106; Abcam, Glostrup, UK; collagen type I: mouse monoclonal IgG, ab90395; Abcam, Milton; fibronectin: rabbit polyclonal IgG, ab6584; Abcam; Ki‐67: Rabbit monoclonal IgG, ab16667; Abcam) diluted in PBS containing 2% BSA for 1 hr at RT (1:100, 1:200, 1:300 and 1:400 respectively). After washing with PBS, cells were incubated for 30 min. at RT with biotinylated secondary antibodies (αSMA: goat‐antimouse IgG2a, 1080‐08; SouthernBiotech, Birmingham, AL, USA; SM22α, Ki‐67 and fibronectin: goat‐anti‐rabbit IgG, E0432; Dako; collagen type I: goat‐antimouse IgG1, 1071‐08; SouthernBiotech) diluted in PBS (1:100) containing 2% BSA for 30 min. at RT. The cells were washed again and incubated with streptavidine‐CY3 (1:100) in PBS containing 1% BSA and (diamidino‐2‐phenylindole) DAPI (1:10,000) for 30 min. After washing with PBS, cell culture wells were mounted with Citifluor and the staining pattern was visualized with fluorescence imaging microscopy (TissueFAXS; TissueGnostics GmbH, Wien, Austria). TissueFAXS data were analysed with the TissueQuest software as described previously 28.