Quantifying Apoptosis in Lung Carcinoma Tissues

Lewis lung carcinoma tumors were collected and 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 five cryosections (6 µm) were assayed for apoptosis by the TUNEL method (DeadEnd Fluorometric TUNEL System), according to the manufacturer’s protocol (46 (link), 47 (link)). Samples were counterstained with DAPI, 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.