N o clear

The label was performed on Automate Discovery XT (Roche Ventana). In all, 5-µm sections of tissue were deparaffinized with Néo Clear (Dako) and two baths of absolute ethanol. Antigenic sites were unmasked with cell conditioning 1 (CC1) pH 8 (Roche). Sections were incubated with an anti-α-SMA antibody (Abcam) 1 h at 37 °C and with an OmniMap anti-rabbit HRP (Multimer; Roche) 16 min for the detection. Sections were incubated in Haematoxylin II (Roche) 16 min and with Bluing reagent (Roche) 4 min. Sections were dehydrated in two baths of absolute ethanol 1 min and xylene substitute Néo Clear (Dako) and mounted in Pertex (Histolab). Sections were scanned and labelling was quantified with CaloPix software.

Colorations were performed on automate Coverstainer (Dako). Sections of tissue were deparaffinized with Néo Clear (Dako) and two baths of absolute ethanol. For HES coloration, 5-µm sections were incubated in the following baths: haematoxylin (Dako) 2 min, tap water 1 min, ethanol 70% 1 min, eosin (Dako) 3 min 30 s, ethanol 96% 1 min and saffron 4 min. For Masson’s trichrome stain 3-µm sections were probed using the Masson’s trichrome kit (Ral Diagnostics) following the manufacturer’s instructions. All sections were dehydrated in three baths of absolute ethanol 1 min and xylene substitute Néo Clear (Dako). Sections were mounted in Pertex (Histolab). They were scanned and labelling was quantified with CaloPix software.