Immunohistochemical staining (IHC)

Paraffin blocks were cut using manual rotary microtome (AccuCut, Sakura, Torrance, USA) to 3–4 µm paraffin sections. Immunohistochemical staining using rabbit anti-CRKL antibody [Y244] monoclonal antibody (1:50; 16 h 4 °C; ab32018; Abcam, Cambridge, UK) was performed according to the protocol described previously and¹⁶. The antibody complex was detected using EnVisionFlex Anti-Mouse/Rabbit HRP-Labeled Polymer (Dako, Agilent Technologies) and localized using 3-3′diaminobenzidine (DAB) as chromogen.

The protein expression was analyzed at 20x original objective magnification using the light microscope ECLIPSE E400 (Nikon Instruments Europe, Amsterdam, Netherlands). The level of CRKL protein was evaluated according to modified immunoreactive scale (IRS) described by Remmele and Stegner¹⁷. In detail, IRS was evaluated as the ratio of the percentage of positive stained cells/area (PP) and the intensity of the color reaction (SI) (IRS = SI × PP) and presented in the 0/+/++/+++ scale (0 no protein, + weak expression; ++ moderate expression; +++ strong expression).