Lightning module

Barley plants cv. Golden Promise grown for seven days on a control or 100 μg/ml zeocin media were used to assess root morphology at microscopic level. Three representative plant samples were chosen for imaging. The whole root was stained by pseudo-Schiff propidium iodide staining as described (Coiro and Truernit⁶⁰ ). Incubation with propidium iodide and Schiff reagent lasted for 48 h. Following the overnight incubation with chloral hydrate solution roots were mounted on glass in water. Imaging was performed using Leica TCS SP8 STED3X confocal microscope (Leica Microsystems, Wetzlar, Germany), equipped with an HC PL APO CS2 10×/0,4 DRY objective, hybrid detectors (HyD), and the Leica Application Suite X (LAS-X) software version 3.5.5 with the Leica Lightning module (Leica, Buffalo Grove, IL, USA). For propidium iodide acquisition, laser excitation at 534 nm and emission at 550–730 nm were used. The maximal projection pictures were constructed from aligned Z-stack images of approximately 250–300 μm steps, containing 45 individual optical sections. The images were post-processed by Leica Lightening software module.

The immunostaining was performed as described (Jasenčáková et al., 2001 (link)). EYFP-FIB1 was detected with primary mouse antisera against FIB1 (1:100; ab4566; Abcam) and secondary antibodies goat anti-mouse-Cy5 (Alexa Fluor^® 647; 1:300; A21235; Invitrogen) or with a goat anti-mouse-Cy3 (Alexa Fluor^® 546; 1:300; A-11003; Invitrogen) for nuclei or metaphase chromosomes, respectively. Alternatively, EYFP-FIB1 on metaphase chromosomes was detected with rabbit antisera against GFP (1,100; ab290; Abcam) recognizing also EYFP and secondary antibodies goat anti-rabbit-Cy3 (Alexa Fluor^® 647; 1:300; A-11010; Invitrogen) for metaphase chromosomes. Nuclei and chromosomes were counterstained with DAPI dihydrochloride (1 μg mL⁻¹) in a Vectashield medium (Vector Laboratories).
Microscopic images were acquired using a Leica TCS SP8 STED3X confocal microscope (Leica Microsystems, Wetzlar, Germany), equipped with an HC PL APO CS2 63 ×/1.40 Oil objective, hybrid detectors, and the LAS-X software version 3.5.5 with the Leica Lightning module (Leica, Buffalo Grove, IL, United States). Confocal images were captured separately in sequential scans, to avoid spectral mixing, using 405 nm (DAPI), 508 nm (EYFP), 557 nm (Alexa Fluor^® 546), and 594 nm (Alexa Fluor^® 647) laser lines for excitation and appropriate emission spectrum. Pictures were processed in Adobe Photoshop version 12.0 (Adobe Systems).