Envisionflex anti mouse rabbit hrp labeled polymer

Prepared slides with tissue sections were deparaffinized and rehydrated. In the first step, antigens were retrieved using a high-pH buffer (Dako, Agilent Technologies, USA) at 95-98°C for 20 min in PT Link (Dako, USA). Then, to block the endogenous peroxidase activity as well as the nonspecific binding sites, the preparations were incubated with 3% H₂O₂ (10 min at room temperature (RT)) and 3% BSA (15 min at RT), respectively. The incubation with the primary rabbit polyclonal anti-NF-κB antibody (1 : 400) and rabbit monoclonal anti-SATB1 antibody (1 : 100) was performed for 30 min at RT. The use of EnVisionFlex+ Anti-Mouse/Rabbit HRP-Labeled Polymer (Dako, Agilent Technologies) for 20 min at RT and 3,3′diaminobenzidine (DAB) enabled the localization of the antigen-antibody complex. In addition, the tissues were counterstained in Mayer's hematoxylin. Finally, tissue sections were dehydrated in ethanol of increasing concentration (from 80% to 98%), then cleared in a series of xylenes (from I to IV), and cover-slipped in a medium (Dako, Agilent Technologies, USA).

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 and^{16 (link)}. 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).