Er2 buffer

4 µm formalin fixed paraffin-embedded (FFPE) sections were cut from tissue blocks and maintained at 60 °C for 2 h. FFPE sections were stained with Cleaved Caspase 3 (CST, 9661) and Ki67 (Abcam, ab16667) antibodies using the Leica Bond Rx autostainer. Sections were loaded onto the autostainer, de-waxed (Leica, AR9222) and epitope retrieval carried out. Both antibodies were retrieved using ER2 buffer (Leica, AR640) for 30 min at 95 °C. Sections were rinsed with Leica wash buffer (Leica, AR9590) before peroxidase block was performed using an Intense R kit (Leica, DS9263). Caspase 3 was stained at a previously optimised dilution of 1/500 and Ki67 at 1/100 before washing with Leica wash buffer and then application of rabbit envision secondary (Agligent, K4003) and visualised using DAB in the Intense R kit. Sections were counterstained with Heamatoxylin and mounted using DPX (CellPath, SEA-1300-00A).
IHC slides were scanned using a Leica SCN 400F scanner at ×20 magnification and images uploaded to Halo Image analysis platform (Indica Labs.). Images were analysed using the CytoNuclear v1.5 algorithm. The percentage of Ki67 or Cleaved Caspase 3 positive cells in prostate tumours or lymph nodes was calculated. Additional measurements such as the average cell area and average cytoplasmic area were also taken.

Antigen retrieval was performed using Er2 buffer (Leica), and sections were incubated with acyl-CoA dehydrogenase (ACAD) (1:500, Servicebio) and BCKDH (1: 500, Servicebio) primary antibodies for 24 h at 4°C. Following incubation with anti-rat-HRP or anti-rabbit-HRP secondary antibodies, the sections were stained with DAB chromogenic substrate. Counterstaining was performed using haematoxylin. Images were obtained using an Eclipse Ti-S fluorescence microscope (Nikon).