Anti dig fitc

A denaturation mixture in volume 25 µL containing 12.5 µL 100% formamide, 5 µL 50% dextran sulphate, 2.5 µL 20× SSC, 3 µL water, and with 1 µL of each probe (26S, 5S) in a final concentration of 100 ng per used volume was denatured at 96 °C for 10 min, then rapidly cooled for 2 min. The mixture was applied onto a chosen slide and co-denatured on a hot plate at 80 °C for 2 min, then incubated overnight at 37 °C in a humid chamber box. Post-hybridization washing was carried out at 42 °C with the following steps: 2× SSC twice for 5 min, 10% formamide in 0.1× SSC twice for 5 min, 2× SSC for 5 min, and 4× SSC with 0.05% Tween-20 for 5 min. Biotin and digoxigenin-labelled probes were immunodetected using streptavidin-Cy3 (GE Healthcare, Buckinghamshire, United Kingdom; 1:750 dilution) and anti-DIG-FITC (Roche; 1:250 dilution) antibodies, respectively. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in Vectashield (Vector Laboratories, Burlingame, CA, USA).
Images were captured using an Olympus BX 51, Olympus, Tokyo, Japan fluorescence microscope equipped with an Olympus DP72 CCD camera. Three greyscale images of each mitosis event were taken, and images were pseudocoloured using Adobe Photoshop CS6 software (chromosomes—blue, 26S rDNA signals—green, 5S rDNA signals—red).

The fixation of the material and the preparation of cytological preparations from the root meristems were performed in accordance with the methodology presented in the article [80 (link)]. The probes were localized on Ps. libanotica and Ps. tauri chromosomes using fluorescent in situ hybridization (FISH) according to the protocol published in [81 (link)]. Detection was carried out using sreptavidin-Cy3 (Vector Laboratories, Peterborough, UK) and Anti-dig-FITC (Roche, Basel, Germany). After the hybridization of the probes, chromosomes were stained with DAPI in the Vectashield medium (Vector Laboratories, Peterborough, UK). The signals were visualized using a DFC 9000 GTC fluorescence microscope (Leica Camera, Wetzlar, Germany) and further processed in Adobe Photoshop (Adobe, Inc., San Jose, CA, USA).

~20-kb FISH probes were generated using a long-range PCR kit (Encyclo Plus PCR (Evrogen)) by PCR-amplification of 4 tiling genome fragments covering either the region 2 L:16964000–16982000 or 2 L:17310000–17328000, with the use of primer pairs provided in the Supplementary Table 1. 1 µg of template DNA for hybridization was labelled by random primed synthesis with the DIG DNA labelling kit (Roche) or by ChromaTide Alexa Fluor 546–14-dUTP (Life Technologies). Probes were further combined and hybridized with S2 cells as described previously^{23 (link)}. For NL or FISH probe detection, as the primary antibodies we used guinea pig polyclonal anti-LBR^{26 (link)} (1:1000), or rabbit polyclonal anti-lamin Dm0^{51 (link)} (1:500) and sheep polyclonal anti-DIG-FITC (1:500, Roche). As the secondary antibodies we used Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Invitrogen), or Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 488-conjugated goat anti-FITC IgG (Invitrogen).