Material:Preparation, Staining and Sampling

Tissue sections:

15 µm thick paraffin sections from routinely processed prostate cancer tissue were deparaffinized and rehydrated.

DNA probes and probe labeling:

â$\alpha$-satellite DNA probes specific for the centromeric region of chromosome 7 were generated from clone 680TA [18]. DNA probe was labeled by nick-translation with biotin-dUTP (Boehringer, Mannheim) according standard procedure [18].

Hybridization:

The fluorescence in situ hybridization (FISH) was performed according to [1]. Briefly, the slides were treated with pronase E (0.05% in 2xSSC, Merck, Darmstadt, FRG) at 37$o$C for 20 to 35 minutes. After denaturation (70% formamide/2xSSC) at 72$o$C for 20 to 45 minutes, the slides were dehydrated in an ethanol series. The hybridization mix consisted of carrier DNA (hering sperm DNA, SIGMA), 55% formamide/1xSSC, 10% dextran sulphate and the labeled DNA probe. This mixture was applied to the slides under a glass cover slip and sealed with rubber cement. After overnight incubation at 37$o$C post-hybridization washes were performed at 43$o$C in three changes of 50% formamide/2xSSC, PN buffer [9], and 3% bovine serum albumin (BSA, SIGMA).

Detection and counter stain:

The biotin-labelled centromere 7 probe was detected by streptavidin-fluorescein isothiocyanat (FITC) and biotinylated anti-streptavidin conjugates (Camon, Wiesbaden). The nuclei were counter stained with propidium iodide (PI, SIGMA) and mounted with an antifade solution as described by [5]. Staining procedure was performed according to [14].

Sampling:

Representative tumor and non-tumor (as control) regions with unambiguous FISH signals and well-preserved nuclei morphology were selected for acquisition by CLSM.