Anti hsp47

Following antibodies were used, mouse monoclonal ROS-BC6 (BC6) (anti-prion) 1.3 mg/ml (TSE Resource Centre, Cat no: RC 063^{74 (link)}), rabbit polyclonal anti-LC3 (MBL, PM036) 1:3000 for WB and 1:300 for imaging, anti-acetyl alpha tubulin (6-11B-1) (Santa Cruz biotech, sc-23950) and mouse monoclonal anti- acetylated tubulin (Lys40) (Proteintech, cat no 66200-1-Ig) both used at 1:1000 for WB and 1:300 for imaging, mouse monoclonal anti-DyneinHC (C-5) (Santa Cruz biotech, sc-514579) 1:1000 for WB, rabbit polyclonal anti-Tubb6 (Aviva systems biology, ARP60382_P050) 1:1000 for WB and 1:300 for imaging, rabbit polyclonal anti-HSP47 (Abcam, ab77609), rabbit polyclonal anti-UbAP2L (Thermo scientific, PA5-29520) both 1:300 for imaging, rabbit monoclonal anti-HDAC6 (Abcam ab133493) used at 1:100 for imaging and rabbit polyclonal anti-beta actin (GeneTex, cat no GTX109639), Rabbit Polyclonal ATG5 (Sigma, A0731), used at 1:1000 for WB, secondary antibodies goat anti rabbit 488 (Thermo Fisher Scientific A11034) and goat anti mouse 647 (Thermo Fisher Scientific A21236) were both used at 1:500 for microscopy.

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.