Metavue

Preparation of slides and hybridization using bacterial artificial chromosome—fluorescent in situ hybridization (BAC-FISH) was carried as described in [53 (link)–55 (link)]. A B. distachyon BAC clone, ABR1-63-E6, that hybridizes all chromosomes of B. distachyon, but not those of B. stacei was labelled by random priming with biotin-14-dUTP (Invitrogen, Life Technologies) [56 (link)]. A ribosomal DNA probe, pTa 71,[57 (link)] which contains a 9-kb EcoRI fragment of rDNA repeat unit (18S-5.8S-26S genes and spacers) isolated from Triticum aestivum was labelled with Alexa-488 dUTP by random priming. Biotinylated probe was immunodetected using Texas Red avidin DCS (Vector Laboratories) and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories) containing 2.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analysed using MetaVue^TM (Universal Imaging Corporation,Downington, PA).

Staining with DAPI (diamidino-2-phenylindole; Sigma–Aldrich, United States) was carried out essentially as described already (Patkar et al., 2010 (link); Ramanujam and Naqvi, 2010 (link)). Bright field and epifluorescence microscopy was performed with an Olympus IX71 or BX51 microscope (Olympus, Tokyo, Japan) using a Plan APO 100X/1.45 or UPlan FLN 60X/1.25 objective and appropriate filter sets. Images were captured with Photometrics CoolSNAP HQ camera (Tucson, AZ, United States) and processed using MetaVue (Universal Imaging, Downingtown, PA, United States), and Adobe Photoshop 7.0.1 (Mountain View, CA, United States). Time-lapse or live cell fluorescence microscopy was performed using a an UltraView RS-3 spinning disk confocal system (PerkinElmer Inc., United States) using a 491 nm 100 mW and a 561 nm 50 mW laser illumination under the control of MetaMorph Premier Software (Ramanujam et al., 2013 (link),BR52). Typically, z-stacks consisted of 0.5 μm-spaced planes for every time point. Image processing and preparation was performed using Fiji², and Adobe Photoshop.

Root tips were collected in 0.04% 8-hydroxyquinoline solution and incubated for 2 h at room temperature, followed by another 2 h at 4 °C. Root tips were then transferred to Carnoy’s I solution (3:1 parts ethanol: acetic acid) and incubated for 24 h before being transferred to 70% ethanol for storage at −20 °C. The procedure for mitosis slide preparation from root tips was as reported by Mason et al. (2014a (link)), using the DAPI as the fluorescent stain. Fluorescence images were captured using a Cool Snap HQ camera (Photometrics) on an Axioplan 2 microscope (Zeiss) and analyzed using the MetaVue (Universal Imaging).

Mammary gland whole mount analysis was carried out as described by Kleinberg et al. [23] (link). All chemicals came from Merck (Darmstadt, Germany), unless otherwise stated. After harvesting, tissues were fixed in ice-cold 4% paraformaldehyde for 2 h. Mammary glands were stored in 70% ethanol until further analysis. In short, following 3 washes with aceton for 30 min, mammary glands were rehydrated in 100% and 95% ethanol (30 min each). The glands were stained for 1 h with filtered hematoxylin, followed by thorough rinsing with tap water. Background staining was minimized by incubation (3×35 min) in a solution of 150 ml 100% ethanol, 150 ml aqua dest. and 7.5 ml HCl 1N (Sigma-Aldrich Chemie GmbH, Munich, Germany). Subsequently tissues were dehydrated in 70%, 95% and 100% ethanol (2×30 min for each step), followed by an incubation in Xylol (Roth, Karlsruhe, Germany) overnight. For long term storage mammary gland whole mounts were placed in methylsalicylate. Digital images of squash preparations were taken by a transmitted-light microscope stand mounted with a NIKON D200 camera (Nikon GmbH, Düsseldorf, Germany) at a distance of 60 cm all in one day. Picture analysis was performed using MetaVue software (MetaVue, Universal Imaging Corp., Downingtown, PA, USA) and ImageJ [34] (link). Pixel size was 11.125 µmx11.125 µm and pixel area was 123.766 µm².

Time-lapse or live-cell fluorescence microscopy was performed using a Zeiss Axiovert 200 M microscope (Plan Apochromat 1006, 1.4NA objective) with an Ultra-View RS-3 spinning disk confocal system (PerkinElmer Inc., Shelton, CT, USA) which was equipped with a CSU21 confocal optical scanner, 12-bit digital cooled Hamamatsu Orca-ER camera (OPELCO, Sterling, VA, USA) and a 491 nm 100 mW and a 561 nm 50 mW laser illumination under the control of MetaMorph Premier Software (Universal Imaging, New York, NY, USA) [39 (link),40 (link)]. Briefly, z-stacks comprised of 0.5 µm-spaced sections that were captured. GFP excitation were performed at 491 nm (Emission 525/40 nm).
Bright-field microscopy was carried out by an Olympus IX71 microscope (Olympus, Tokyo, Japan) equipped with a Plan APO 100X/1.45 objective. Images were captured by Photometrics CoolSNAP HQ camera (Tucson, AZ, USA) and processed with MetaVue (Universal Imaging, Downingtown, PA, USA), Adobe Illustrator (Adobe Inc., San Jose, CA, USA), and ImageJ (LOCI, University of Wisconsin, Madison, WI, USA).

Root tips were collected in 0.04% 8-hydroxyquinoline solution and incubated for 2 h at room temperature, followed by another 2 h at 4 °C. Root tips were then transferred to Carnoy’s I solution (3:1 parts ethanol: acetic acid) and incubated for 24 h before being transferred to 70% ethanol for storage at −20 °C. The procedure for mitosis slide preparation from root tips was as reported by Mason et al. (2014a (link)), using the DAPI as the fluorescent stain. Fluorescence images were captured using a Cool Snap HQ camera (Photometrics) on an Axioplan 2 microscope (Zeiss) and analyzed using the MetaVue (Universal Imaging).