Evaluating PTEN Expression by IHC

We have recently designed and validated a protocol for evaluating PTEN expression by immunohistochemistry (IHC).^{20 (link)} ERG–PTEN dual IHC was performed using anti-ERG (EPR3864) rabbit monoclonal primary antibody (1:100; Cat no. 790-4576, Ventana Medical Systems, Tucson, AZ, USA) and a rabbit monoclonal primary antibody against PTEN (1:25; 138G6- Cell Signaling Technology, Danvers, MA, USA). Dual IHC was performed using an automated protocol developed for the DISCOVERY XT automated slide staining system (Ventana Medical Systems) using UltraMap antirabbit HRP (Cat no. 760-4315, Ventana Medical Systems) for ERG and UltraMap anti-rabbit AP (Cat no. 760-4314, Ventana Medical Systems) for PTEN as secondary antibodies and were detected using ChromoMap DAB (Cat no. 760-159, Ventana Medical Systems) and ChromoMap Blue (Cat no. 760-161, Ventana Medical Systems) for ERG and PTEN, respectively. Nuclear Fast Red counterstain (Cat no. 780-2186 Ventana Medical Systems) was used as the counterstain. Hematoxylin II (Cat no. 790-2208 Ventana-Roche) was used as counterstain. Examples of ERG/PTEN staining in prostate cancer are shown in Figure 2.