In situ hybridization was carried out by incubating the slides with RNase-treated and denatured metaphase spreads at 42°C for 16 hours with approximately 1 µg biotinylated probe in 30 µl of hybrudization solution containing 45 % formamide, 2 x SSC, 10% dextran sulfate amd 10 µg sheared and denatured salmons sperm DNA. The hybridization mix was supplemented with 8 µg sheared genomic mink DNA and incubated efter denaturation for 30 min. at 37°C.
After hybridization, the slides were washed two times in 45% formamide, 2 x SSC and three times in 2 x SSC at 42°C. Visualization of the biotinylated probe was achieved with fluorescein isothiocyanate (FITC)conjugated to avidin, and the signal was amplified with one layer of biotinylated anti-avidin by antibody (Vector Laboratories). The RBP-band pattern generated by propidium iodide in alkaline DABCO antifade solution (Lemieux et al. 1992).