Histological sections (5 μm thick) were cut from te paraffin-embedded skin and vein samples and used for detection of different stromal cell types using specific fluorescent markers, as follows: FITC-labelled avidin (1:400) for mast cells [29 (link)]; FITC-labelled UEA lectin (1:10) for blood vessels [30 (link)]; rabbit polyclonal anti-HSP47 (1:50 Abcam, Milan, Italy) followed by FITC-labelled goat anti-rabbit antibodies (1:32 Abcam) for activated fibroblasts [31 (link)]; and goat polyclonal anti-α-SMA (1:400 Abcam) followed by FITC-labelled rabbit anti-goat antibodies (1:175 Abcam) for both blood vessels and myofibroblasts [32 (link)]. In some slides, nuclei were counterstained in red with propidium iodide. Before each immunolabeling, antigen retrieval was performed using 0.1 M citrate buffer at 96 °C for 10 min. The fluorescent markers and the primary antisera were applied overnight at 4 °C, and the secondary antisera for 2 h at 37 °C. Omission of the primary antibody was used as a negative control for the immunofluorescence reactions. The labelled sections were viewed and photographed using a Zeiss Axioskop UV microscope equipped with a digital camera and Axiovision 4 software (Zeiss, Jena, Germany) or a Leica TCS SP5 confocal microscope. Unless otherwise stated, all reagents were from Sigma-Aldrich.
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