Chromogenic Staining

It was performed on tissue sections using the ChromoMap DAB detection Kit (Inhibitor CM, DAB CM, H2O2 CM, Copper CM) (Ref. 750-159, Ventana Medical System, Tucson, AZ). Briefly, the slides after the melting step of paraffin, were followed by a pre-treatment heat-induced epitope retrieval at 95°C. Then, they were incubated with an inhibitor solution for 4 min to quench endogenous peroxidases activity and the Ventana dilution buffer (Ref. ADB250, from Roche) was used as a blocker for unspecific binding, for 20 min incubation at RT. Next, the slides were incubated with primary antibodies, applied either manually or with ready to use Roche Ventana dispensers at 37°C, for the validated optimum time. This was followed by incubation with horseradish peroxidase (HRP)-conjugated mouse or rabbit secondary antibodies, DAB revelation and hematoxylin counterstaining (Roche, Ref. 790-220B) for 8 min. Then, the slides were washed and dehydrated through immersion in successive baths (2 min each) with an ascending series of ethanol, followed by increasing concentration of xylene before coverslip mounting. The slides were finally scanned by the whole-slide scanner NanoZoomerS60, Hamamatsu (serial # C13210-01, Hamamatsu, Hamamatsu City, Japan).