FISH Procedure

Before FISH procedure, chromosome slides were pre-treated with 1 mg/ml RNase A (Roche) in 2× SSC at 37°C for 1 h and then washed three times for 10 min in 2× SSC. The slides were dehydrated in a series of 70, 85, and 96% ethanol solutions and then air dried. The hybridization mixture (15 μl) containing 40 ng of each labeled probe was added to each slide. Coverslips were placed on the slides and sealed with rubber cement. Slides with DNA probes were co-denatured at 74°Ñ for 5 min, placed in a moisture chamber, and hybridized overnight at 37°C. After removing the coverslips, the slides were washed twice with 0.1× SSC at 44°C for 10 min, followed by two washes with 2× SSC at 44°C for 5 min and the final 5 min wash in 2× SSC at room temperature. Prior to detection, the slides were soaked in 4× SSCT (0.1% Tween-20 in 4× SSC) at room temperature for 3 min and then incubated in a detecting buffer (5% fat-free dry milk in 4× SSCT) at 37°C for 30 min. The slides were washed in 4× SSCT at room temperature for 3 min. In the case of the conserved domain B the CesA-6 subunit was labeled directly by Fluorescein Labeling Kit (Kreatech Biotechnology, Amsterdam, Netherlands), the fluorescent signal amplification using FITC-Alexa 488 antibodies (VectorLabs, Youngstown, OH, United States) was performed.

After incubation for 60 min at 37°C with the detection mixture, the slides were washed three times with 4× SSCT for 3 min each at room temperature, followed by a short rinse in PBS. The slides were dehydrated and air dried in the dark.