Rabbit polyclonal antibody specific for PRR [ref. HPA003156; Sigma-Aldrich (Saint Louis, MO, USA) at 1/50 dilution] was used for the immunostaining of formalin-fixed and paraffin-embedded tumour tissues. The antibody’s specificity was tested previously [27 (link)] and the immunostaining process was performed following routine methods in an automatic immunostainer (Dako Autostainer Plus, Dako-Agilent, Santa Clara, California, USA). Briefly, antigen retrieval was carried out in a low-pH buffer (K8005, Dako, Santa Clara, California, USA) for 20 min at 95 °C. The samples were incubated with the primary antibody for 50 min at room temperature. Then, the primary antibody was washed and samples were incubated for 20 min with secondary anti-rabbit antibody (K8021, Dako). EnVision-Flex detection system together with an HRP-enzyme-labelled polymer (SM802, Dako) was employed. A positive reaction was visualized with diaminobenzydine (DAB) solution (DM827, Dako), followed by counterstaining with haematoxylin (K8008, Dako).
Slides were reviewed under light microscopy for staining evaluation. Two observers independently evaluated the slides and, in the event of discrepancies, samples were re-evaluated to arrive at a final conclusion. As previously reported [27 (link)], staining patterns were scored as negative, weak and intense cytoplasmic and membranous staining.
Slides were reviewed under light microscopy for staining evaluation. Two observers independently evaluated the slides and, in the event of discrepancies, samples were re-evaluated to arrive at a final conclusion. As previously reported [27 (link)], staining patterns were scored as negative, weak and intense cytoplasmic and membranous staining.