Trypsin

Sections were deparaffinized in xylene for 3 × 10 min and rehydrated in 100% ethanol for 2 × 5 min, 95% ethanol for 1 × 5 min, 70% ethanol for 1 × 5 min, and (Phosphate Buffered Saline) PBS for 2 × 5 min. Antigen retrieval was performed with 0.05% trypsin (Zymed Laboratories, Invitrogen) for 10 min at 37 °C. Tissue was blocked for 1 h with 10% BSA before incubation with goat anti-FHL-1 (Abcam, Cambridge, UK; ab23937; 1:250 diluted) or rabbit anti-PCNA (Santa Cruz Biotechnology Inc., Heidelberg, Germany; sc7907; 1:100 diluted) antibodies followed by ImmPRESS anti-Goat or anti-Rabbit Ig (peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA). Negative control experiments were performed with an isotype control (matched host species and isotype) antibody.

DNA fragmentation coupled to the apoptotic response was detected in morphologically identifiable nuclei and apoptotic bodies present in formalin-fixed paraffin-embedded tumors by the TUNEL assay (ApopTag, Chemicon, Temecula, California) following the manufacturer's instructions. Tissue slides were pre-treated with 0.5% trypsin for 15 minutes (Zymed, San Francisco, California), to improve the exposure of DNA.