Fluorescence in situ Hybridization of Plant Nuclei

Flower buds were sampled from 5- to 6-week-old plants and fixed in Farmer’s solution (acetic acid:ethanol, 1:3) for 1 h at 25 °C. Fixed buds were washed with 70% ethanol and then with distilled water for 5 min. The buds were treated with an enzyme solution (2% w/v cellulose Onozuka RS, 0.5% w/v pectolyase Y-23, 10 mM citrate buffer, pH 4.5) at 37 °C for 1 h. The buds were broken by pipetting and then filtered through a 100-µm nylon mesh. The filtered nuclear solution was centrifuged at 5000 × g for 1 min and the pellet was resuspended in Farmer’s solution. A drop of the nuclear solution was placed on a glass slide and allowed to dry. Then, FISH was performed as previously described^{46 (link)}. A centromere probe was amplified by PCR and labeled by nick translation using DIG-Nick Translation Mix (Sigma-Aldrich, St. Louis, MO, USA). Hybridized probes were visualized using a rhodamine-conjugated anti-digoxigenin antibody (Sigma-Aldrich). The number, size, and fluorescence intensity of FISH signals were measured using ImageJ 1.51 g. A binary image was generated from the fluorescent image and “Analyze Particles” in ImageJ was used to determine the signal size. To measure the fluorescence intensity, one pericentromeric signal was clipped from the raw image and “Measure” was used to determine the signal intensity. Subsequently, the background was subtracted from the signal intensity.