2 solution diaminobenzidine dab kit

The processing of tumour samples was performed in accordance with previously established protocols.²⁷, ²⁸ Normal horse serum (Jackson ImmunoResearch, West Grove, PA, USA) was applied for 60 minutes to block non‐specific epitopes. This was followed by incubation of the sections at 4°C overnight with primary antibodies. For immunohistochemistry, the 2‐Solution diaminobenzidine (DAB) kit (Invitrogen, Waltham, MA, USA) was utilized to detect signals following a 60 minutes incubation with suitable secondary antibodies conjugated to HRP (Bio‐Rad, Hercules, CA, USA) at room temperature. This was followed by haematoxylin counterstaining and mounting in Acrymount (StatLab). The staining was evaluated at ×400 magnification in a minimum of three areas and based on the scores, were categorized per specimen as follows: score 0, negative; score 1, <25% positive cells; score 2, 25%‐50% positive cells; score 3, 50%‐75% positive cells; and score 4, >75% positive cells. This was followed by scoring of immunostaining intensity as follows: 1+, weak; 2+, moderate; and 3+, intense. A total score was obtained by multiplying the two above‐mentioned values. Two experienced investigators blinded to the experimental groups and to each other evaluated the immunostaining independently.