Detection of Human Cells in Paraffin Tissues

The detection of human cells within the resected paraffin-embedded tumour tissues was performed as described previously [21 (link)]. In brief, deparaffinised and rehydrated sections were digested for antigen retrieval with 5 µg/mL proteinase K (Roche Diagnostics, Mannheim, Germany) for 15 min at 37 °C. Prehybridisation was completed at 42 °C for 1 h in a hybridisation buffer consisting of 4 × SSC (saline sodium citrate), 50% deionised formamide, 1 × Denhardt’s solution, 5% dextran sulfate and 100 µg/mL salmon sperm DNA. Samples were further incubated for 16 h at 42 °C with fresh hybridisation buffer supplemented with 0.2 ng/µL heat-denatured digoxigenin-labelled ALU probe, which was synthesised by PCR as described previously [19 (link)]. Detection was performed using anti-Digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche Diagnostics) and NBT/BCIP (Linaris) as substrate. Sections were counterstained with Methyl Green (Linaris), mounted with Neomount (Merck-Millipore, Burlington, MA, USA) and imaged using a Keyence BZ-X800 microscope (Keyence).