Envision peroxidase detection system

Immunohistochemical expression of c-Myc (Y69, 1:300, Epitomics, Burlingame) and beta-tubulin-3 (3F3, 1:40000, Seven Hills, Bioreagents, Cincinnati) was evaluated. All the sections were developed with the Envision peroxidase detection system (DAKO, Carpinteria, CA), a technique very sensitive and capable of preventing false positivity due to endogenous biotin present in the tissues. The c-Myc was considered positive when expressed at the nuclear level and classified into three groups: 0 (negative), 1 + (>15% weakly positive cells), 2 + (>15% strongly positive cells) (Fig 1). The beta-tubulin-3 was considered positive when expressed at the level membranous and cytoplasmic as follows: 0 (negative), 1 + (>15% weakly positive cells), 2 + (>15% strongly positive cells) (Fig 2). For beta-tubulin-3, we have also reported a positive score in the reactive tissue adjacent to the tumor. In order to simplify the results and choose a single representative value for each variable, we reported the median of the number of observations corresponding to each patient. The median score per case was used for the statistical analysis.

The immunohistochemistry was performed using specific antibodies against mouse monoclonal Androgen Receptor (AR, clone 2F12, dilution 1:25, Novocastra, Dublin, OH, USA) [27 (link)], mouse monoclonal Cytokeratin 5/6 (CK5/6, Clone CK5/6.007, dilution 1:100, Biocare Medical, Concord, CA, USA) [28 (link)], mouse monoclonal p-AKT (Clone HP18, dilution 1:75, Novocastra), [29 (link)], rabbit monoclonal p-p44/42 MAPK (Clone 20G11, dilution 1:100, Cell Signaling Technology, Boston, MA, USA), [30 (link)] and mouse monoclonal PTEN (Clone 6M2.1, dilution 1:200, DakoCytomation, Glostrup, Denmark) [31 (link)]. Immunoreactions were obtained by incubating sections with specific primary antibodies for 15 minutes. Immunodetection was performed using a non-biotin highly sensitive system (EnVision Peroxidase Detection System, Dako), preventing possible false-positive staining due to endogenous biotin present in the tissue. The slides were then incubated with substrate chromogen solution diaminobenzidine (DAB) for 10 minutes and counterstained with haematoxylin. Specifically, mouse monoclonal EGFR (Clone 2-18C9) immunoreaction was executed using EGFR pharmDx™ Kit (DakoCytomation) [32 (link)], according to manufacturer’s instructions.