Immunohistochemical and immunofluorescence analyses

Tumor samples were fixed with 10% neutral buffered formalin and embedded into paraffin or fixed with 1% PFA and frozen in a cryo-embedding medium (OCT, Bio-Optica). Slides were cut and stained with Hematoxylin (Bio-Optica) and Eosin (Bio-Optica) for histological examination. Slides were then deparaffinized, serially rehydrated and stained with mouse monoclonal anti-PCNA (Dako), rabbit polyclonal anti-Caspase3 (R&D System), rat monoclonal anti-CD31 (Dianova), followed by the appropriate secondary antibodies. Immunoreactive antigens were detected using streptavidin peroxidase (ThermoScientific) and the DAB Chromogen System (Dako) or alkaline phosphatase-conjugated streptavidin (ThermoScientific) and Vulcan fast red Chromogen (Biocare Medical). After chromogen incubation, slides were counterstained with Hematoxylin (Bio-Optica) and images were acquired by a Leica DMRD optical microscope (Leica). The percentage of PCNA positive cells was determined on digital images of 5–7 tumors per group (2–4 X 200 microscopic fields per sample); clear red nuclei were regarded as positive cells and were selected with the magic wand tool of Photoshop. For each field, the number of positive cell pixels indicated in the histogram window was reported as % on the number of negative cell pixels (PCNA index).

For immunofluorescence evaluation of frozen samples, sections were air-dried, fixed in ice-cold acetone for 10 min and incubated with a Rabbit polyclonal anti-Neutrophil Elastase antibody (Abcam), followed by a secondary antibody conjugated with Alexa 488 (Life Technologies). Image acquisition was performed using a Zeiss LSM 800 confocal microscope.