Immunofluorescence Staining for Protein Detection

Immunofluorescence staining was performed as previously described (26 (link), 56 (link)). Briefly, deparaffinized 4–5 μm thick sections were boiled at 85°C to 90°C for 60 minutes with 0.05% citraconic acid solution (ImmunoSaver; Wako) to retrieve antigens. After blocking for 60 minutes (Blocking One Histo, Nacalai Tesque), the sections were incubated overnight at 4°C with primary antibodies diluted in immune reaction enhancer solution (Can Get Signal) at 1:50 (anti-hmUhrf1), 1:100 (anti-hUHRF1, mPdpn, hPDPN, mFap, hFAP, Thy-1, mCD45, hCD45, F4/80, CD3, cleaved-caspase-3, Ki67), or 1:500 (anti-GFP). After washing with PBS, 5 μg/mL secondary antibodies with DAPI were reacted for 60 minutes at room temperature. To detect apoptotic cells histologically, TUNEL was performed (Roche). After blocking, deparaffinized sections were reacted with fluorescein-conjugated dUTP for 60 minutes at room temperature according to the manufacturer’s instructions.