Coolsnap videocamera

Tissues were fixed in 10% (v/v) phosphate-buffered formalin according to standard procedures,^{16 (link)} paraffin-embedded and cut into consecutive 2.5–3 μm sections that were then stained either with either hematoxylin–eosin and Gomori silver (MicroStain MicroKit, Bologna, Italy), or immunostained with anti-transforming growth factor (TGF)-β1 (sc-146, Santa Cruz Biotechnology) antibodies. Immunoreactions were detected with STAT-Q Peroxidase–AEC Staining System Kit (Innovex Biosciences, Richmond, CA, USA). Images acquired with the Light microscope (ZEISS, Axioskop, Germany) equipped with a Coolsnap Videocamera were quantified with the MetaMorph 6.1 Software (Universal Imaging Corp., Downingtown, PA, USA). Electron microscopyand immuno-electron microscopy observations were performed as described previously.^{16 (link), 17 (link)}

Cytocentrifuged cells were fixed with 3.7% paraformaldehyde, blocked with 10% normal donkey serum. Samples were then incubated with the following primary antibodies (working diluition, 1:500): NF-κB, pCREB, ATF2, cyclin D1 (Cell Signaling Technology); ATF3 and cyclin A1 (Santa Cruz Biotechnology). Samples were then incubated with IgG-FITC and IgG-TRITC (working dilution, 1:100) (Jackson ImmunoResearch, West Grove, PA, USA) as secondary antibodies. The nuclei were counterstained with DAPI (Vector Laboratories, Inc., Burlingame, CA). All observations were performed using a Zeiss Axioscope light microscope equipped with a Coolsnap Videocamera to acquire images to analyze with MetaMorph 6.1 software (Universal Imaging Corp, Downingtown, PA, USA).