Fluorescence In-situ Hybridization (FISH)

FISH was performed at two locations, using different protocols. At the Rizzoli Orthopedic Institute, FISH was performed using the LSI FOXO1 Dual Color Break-apart DNA probe (13q14) (Abbott Molecular, Des Plaines, IL, USA) and the SPEC MDM2/CEN 12 Dual Color probe (12q14.3–12q15) (ZytoVision GmbH, Bremerhaven, Germany) according to the manufacturer’s protocol. Tissue sections of 4 μm were mounted on positively charged slides (Dako, Glostrup, Denmark). Slides were heated overnight (60°C), dewaxed in xylene, and treated with an ethanol-to-water series. This was followed by incubation in TE solution (TRIS 5 mM-EDTA 1 mM) at 96°C for 15 minutes, rinsed in distilled water, and digested with pepsin (0.04%) in 0.01N HCl at 37°C for 5 to 15 minutes, then washed again in distilled water. Slides were finally dehydrated in ethanol (96%) and air dried. Next, the probes were applied to the target area and the slides were coverslipped and sealed with rubber cement. The samples and probes were co-denaturated in Dako Hybridizer (Dako, Glostrup, Denmark) at 85°C for 1 minute and incubated overnight at 37°C. The following day, the coverslips were removed and the slides were washed 2 min at 73°C in 0,4 X SSC/0.3%NP40 and 1 min at room temperature in 2 X SSC/0.1% NP40. The slides were then left to dry in the dark at room temperature; the nuclei were subsequently counterstained in Vectashield Antifade solution with DAPI (Vector Laboratories, Inc. Burlingame CA, USA). Fluorescence signals were counted using an OLYMPUS BX41 fluorescence microscope (Olympus, Hamburg Germany), at 100X under oil immersion using an appropriate filter set. A minimum of 100 tumor cell nuclei with intact morphology, as determined by DAPI counterstaining, were counted in the previously marked neoplastic area. A positive result was defined as the presence of a visible red and green signals in more than 10% of the cells.

At the University of Manitoba, slides were deparaffinized and dehydrated in 100% ethanol, then incubated in 1 M NaSCN for 30 min at 80°C in a water bath. After rinse in double distilled water, slides were incubated in 3.7% buffered formalin solution (Sigma-Aldrich, Oakville, ON, Canada) in 2x SSC buffer (pH 7.6) for 10 min at RT and washed two times in 2xSSC for 5 min each. Totally 50 µg/ml pepsin was added to 0.01 M 37°C prewarmed HCL and slides were incubated for 6 min followed by two washes in 2xSSC for 5 min each. After exposure to 3.7% buffered formalin solution (Sigma-Aldrich, Oakville, ON, Canada) in 2x SSC buffer (pH 7.6) for 10 min at RT and two washes in 2xSSC, tissues were dehydrated in ethanol. 5 µl of Cy3-labeled peptide nucleic acid telomere (PNA) probe (Dako, Glostrup,Denmark) was applied to each slide to detect (T2AG3)n repeats. Slides and probe were incubated at 80°C for 3 min to denature DNA, followed by hybridization at 30°C for 2 hr using the Hybrite chamber (Vysis, Abbott Diagnostics Mississauga, ON, Canada). Next, slides were washed twice in 70% formamide in 10 mM Tris (pH 7.4) followed by successive washings in PBS at RT for 1 min, in 0.13 SSC at 55°C for 5 minutes and in 2x SSC in 0.05% Tween 20 twice at RT. Slides were counterstained with 40,6-diamino-2-phenylindole (DAPI, Invitrogen, Burlington, ON, Canada) (0.1 µl/ml), washed in doubled distilled water and dehydrated in ethanol. Before imaging, slides were mounted to coverslips using Vestashield antifade mounting medium (Vector Laboratories, Burlington, ON, Canada).

Fluorescent images were captured using the AxioImager Z1 microscope, an AxioCamMR3 camera and a Plan-Apochromat 63x/1.40 Oil DIC M27 lens (all from Carl Zeiss, Canada). Acquisition time was 300 msec for Cy3 (telomeres signals) and 100 msec for DAPI. For each nucleus, images were acquired from 60 optical planes (z-stacks) through the nucleus with a sampling distance in the xy axis of 107 nm and z axis of 200 nm using ZEN 2 blue edition software (Carl Zeiss, Canada). Images were deconvolved using a constrained iterative algorithm (Schaefer et al., 2001) available in the ZEN software (Zeiss).