Application suite x 2

Tumor paraffin sections were deparaffined and probed using an anti-PCNA antibody (1:300 dilution in PBS 0.1% 24:6 BSA; Santa Cruz Biotechnology). Secondary antibody was a goat anti-mouse horseradish peroxidase (HPR)-conjugated antibody (1:300 dilution in PBS 0.1% BSA; Thermo Fisher Scientific). DAB (Dako, Agilent Technologies) was used as substrate of HPR conjugated to secondary antibody. To count PCNA-positive cells, pictures were acquired by digital camera suite Leica Application Suite X 2.0.0.14332 (Leica Microsystems). The ten fields with the highest density of positive nuclei were captured at ×200 final magnification and positive vs total cells were counted using ImageJ software. Only nuclei with a strongly positive label were counted.

Apoptosis was evaluated by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay (ApopTag Plus, Millipore). Briefly, paraffin embedded slices of tumors were deparaffined and TUNEL assay was performed following the manufacturer's instruction. Positive cells were stained by a brown precipitate of oxidized 3,3′-diaminobenzidine (DAB) (Dako, Agilent Technologies). Slides were observed under optical microscope Olympus AX70 PROVIS (Olympus). To count positive cells, pictures were acquired by digital camera suite Leica Application Suite X 2.0.0.14332 (Leica Microsystems, Wetzlar, Germany). After excluding areas of necrosis, the number of apoptotic cells per field was counted in 10 randomly chosen sections using ×200 final magnification.

Collagen fibers in hearts were detected by Masson's trichrome stain kit (Bio-Optica Milano S.p.a., Milan, Italy). Briefly, paraffin-embedded slices of hearts were deparaffined and trichrome stain was performed following the manufacturer's instruction. Collagen fibers were stained by aniline blue solution included in the kit. Slides were observed under optical microscope Olympus AX70 PROVIS (Olympus). Pictures were acquired by digital camera suite Leica Application Suite X 2.0.0.14332 (Leica Microsystems). The percentage of blue area vs total area was counted in ten randomly chosen fields at ×200 final magnification using ImageJ software.