K8004

Samples (4 µm sections) from paraffin-embedded tissue blocks were pretreated in target retrieval solution (pH = 9), Dako (K8004) in PT Link (Dako, Oslo, Norway) for 20 min at 97 °C. Subsequently, the samples were incubated with antibody from Abcam (ab50276) against PRL-3 (diluted 1:300) for 40 min in room temperature. The detection system used was EnVision/HRP Rabbit, Dako (K4011).

4 μm thick PRCC sections were firstly de-waxed and pretreated in high pH antigen retrieval solution (Tris-EDTA, pH = 9.0; K8004, DAKO) at 95°C for 30 minutes. Sections were then treated with protease 3 (760–2020, Ventana Medical Systems) for 6 minutes and then incubated in CONFIRM anti-total c-MET (SP44) rabbit monoclonal antibody for 1 hour. Goat anti-rabbit IgG FITC (5ug/ml, ab6717, Abcam) was added and sections were incubated for 30 minutes at room temperature. After post-IF fixation in neutrally-buffered formalin for 10 minutes, sections were dehydrated in ethanol series and air-dried. Probes for MET gene and CEP7 were applied and slides were co-denatured at 79°C for 6 minutes, and then incubated at 37°C for more than 30 hours. After overnight incubation, slides were washed with 0.3% NP40/2 x SSC (pH 7.0–7.5) at 72°C for 2 minutes, and then in 2 x SSC at room temperature for 2 minutes. Slides were finally dehydrated in ethanol series, air-dried, and mounted with mounting medium with DAPI (Cat #H-1200, Vector Labs). Slides were analyzed using fluorescent microscopy.

IHC analysis was performed on a 4 μm thick TMA. MET immunostaining was performed using CONFIRM anti-total c-MET (SP44) rabbit monoclonal antibody (790–4430, Ventana Medical Systems) on an automatic immunostainer (Discovery XT, Ventana Medical Systems) following standard staining protocol from Ventana Medical Systems. Ki67 IHC was performed as follows: slides were dried at 37°C overnight and then baked at 56°C for 30min, then deparaffinized in xylene and rehydrated through a graded series of ethanol concentrations. Antigen retrieval was performed in PTlink using high pH target retrieval solution at 97°C, 15 minutes (Target Retrieval Solution, pH 9, K8004, DAKO). Slides were covered with primary antibody solution (M7240, DAKO) and incubated at room temperature for 60 minutes, then washed twice in TBS-T and incubated with the EnVision+ system-HRP labelled polymer anti-rabbit secondary antibody (K4003, DAKO) for 30 minutes. Following two additional washes in TBS-T, slides were visualized using DAB substrate-chromagen (K3468, DAKO). All slides were independently evaluated and results were agreed by two pathologists who were blinded to patient information.