TUNEL assay

The collected melanoma transplants were fixed in ice cold 4% paraformaldehyde before being rinsed in PBS and cryo-protected overnight in 30% sucrose. Tissues were then embedded in O.C.T. Compound (Sakura, AJAlphen aan den Rijn, The Netherlands) and cut in a CM1850 UV cryostat (Leica Biosystems, Wetzlar, Germany). At least 5 cryosections (6 μm) were obtained from each tumour and assayed for apoptosis by the TUNEL method (DeadEnd Fluorometric TUNEL System; Promega, Milano, Italy), according to the manufacturer's protocol. After DAPI counterstaining, samples were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) and examined using a DMI4000 B automated inverted microscope equipped with a DCF310 digital camera (Leica Microsystems, Wetzlar, Germany). Image acquisition was controlled by the Leica LAS AF software.