K346430

Paraffin-embedded specimens from patients with primary and relapsed castration-resistant PCa that had been collected between 1990 and 2010 at Mackay Memorial Hospital were included in this study. Ethics was approved by the Mackay Memorial Hospital Institute Review Board. Informed consent was written. Tissue sections were deparaffinized in xylene, rehydrated through graded ethanol (100, 90, 80, 70, 50%), subjected to antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0) for 10 min, blocked for endogenous peroxidase activity with 3% H₂O₂ for 10 min at room temperature, and then blocked with antibody diluent containing background-reducing components (DAKO, S302281). Finally, the slides were stained overnight at 4°C with beclin1 (GeneTex, GTX61619), LC3 (Novus Biologicals, NB110-57179), REST (BETHYL, IHC-00141), or CgA (abcam, ab15160) specific antibodies and visualized following standard protocol using 3-amino-9-ethylcarbazole (AEC) as chromogen (DAKO, K346430) in the presence of hematoxylin counterstaining. The histology and staining was evaluated by a pathologist in a blind fashion using a four-tier scale representing negative (0), weak (1), intermediate (2), and strong (3) signals.

Paraffin-embedded specimens from patients with primary PCa and CRPC that was collected at Taipei Veterans General Hospital (TVGH) were included in this study. Ethics was approved by the TVGC Institute Review Board. Written informed consent was obtained. Tissue sections were deparaffinized in xylene, rehydrated through graded ethanol, antigen was retrieved by microwaving sections in 10 mM citrate buffer (pH 6.0) for 10 minutes, and endogenous peroxidase activity was inactivated with 3% H₂O₂ for 10 minutes at RT. Slides were blocked with antibody diluent containing background-reducing components (DAKO, S302281) and then stained overnight at 4 °C with anti-REST (1:200, BETHYL). anti-MAOA (1:200, Abcam) and cytokeratin 5 (1:200, Genetex) antibodies and visualized following standard protocol by using 3-amino-9-ethylcarbazole (AEC) as chromogen (DAKO, K346430) in the presence of hematoxylin counterstaining. Scoring of REST and MAOA expression in human PCa samples was performed by a pathologist (Dr. Pan) and was based on H-score, which is derived by multiplying the staining intensity (0–4) with the percentage of epithelial cells with positive staining.