Dmi4000 b automated inverted microscope

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.

Using published protocols [106 (link),108 (link)], MCF7 or U373 cells cultured in 120-mm coverslips were fixed in 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 10 min. Cells were pre-incubated for 1 h min with 5% of normal goat serum (Life Technologies, Monza, Italy) in 0.1 M PB (pH 7.4) containing 0.1% Triton X-100, before overnight incubation with the rabbit monoclonal anti-cleaved caspase 7 (Cell Signaling Technology, Danvers, MA, USA). In double-label immunofluorescence experiments, the mouse monoclonal anti-cytochrome c primary antibody (Cell Signaling Technology) was used in conjunction with the rabbit monoclonal primary antibody directed to COX IV (Cell Signaling Technology). For fluorescence detection, coverslips were stained with the appropriate Alexa Fluor secondary antibodies (Life Technologies) and mounted on glass slides in a ProLong Gold Antifade Mountant (Life Technologies). DAPI and/or fluorescein phalloidin (cytoskeleton detection) staining was also used. Cells were analysed with a DMI4000 B automated inverted microscope equipped with a DCF310 digital camera (Leica Microsystems, Wetzlar, Germany). When indicated, confocal imaging was performed with a TCS SP8 System (Leica Microsystems). Image acquisitions were controlled by the Leica Application Suite X.

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.