Dako real envision hrp

All specimens were embedded in optimal cutting temperature compound (Sakura Tissue-Tek Xpress, Torrance, CA, USA). Immunohistochemistry was performed on 5 µm thick sections obtained from 4% paraformaldehyde-fixed, paraffin wax-embedded tissue. Following dewaxing and rehydration steps, antigen retrieval was performed using citrate buffer. Sections were blocked with peroxidase blocking solution (Dako, Santa Clara, CA, USA) for 15 min. The sections were incubated with Cdc42 antibody (1:1000, Abcam), Rac1 antibody (1:1000, Invitrogen), and RhoA antibody (1:2000, Novus Biologicals) overnight at 4 °C. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with Dako REAL™ EnVision/HRP (DAKO, Carpinteria, CA, USA) at room temperature for 2 h, and were visualized with substrate-chromogen solution. Counterstaining was performed with Mayer’s hematoxylin (Dako). Stained tissue samples were observed using a Leica light microscope DMI 5000B (Leica, Wetzlar, Germany).

Establishment of the consecutive RCC tissue microarray (TMA) consisting of 453 FFPE tumor samples of different subtypes, the associated patient and tumor characteristics and the procedure of immunohistochemical analysis have been recently described in detail^{45 (link)}. Immunohistochemical staining (IHC) was performed by applying a 1:50 dilution of a rabbit monoclonal panCREB1-specific antibody (clone 48H2, #9197 Cell Signaling Technology) to 5 µM sections of the TMA. An HRP-linked polymer containing anti-rabbit (and anti-mouse) secondary antibody (Dako REAL™ EnVision™/HRP, Dako, Denmark) was employed and detection was visualized using 3,3′-diaminobenzidine chromogen substrate (Dako REAL™ DAB + Chromogen, Dako, Denmark). Staining evaluation and scoring was performed by two experienced observers (AH and CS). Staining intensity of each tissue core was recorded as 0 (absent), 1 (weak), 2 (moderate) or 3 (high). Simultaneously, the percentage of stained tumor cells was recorded as 0 (no stained tumor cells), 1 (1–9% stained tumor cells), 2 (10–50% stained tumor cells), 3 (51–80% stained tumor cells) or 4 (81–100% stained tumor cells). The intensity score of each sample was then multiplied by the percentage score it had achieved, i.e. values between 0 and 12 were possible. Finally, these results were grouped to negative (scores 0–2) and positive (scores 3–12).