Cellsens dimension

FISH was performed as described previously15 (link)27 (link). Briefly, 10–40 pmol of primary c2ca-amplified Oligopaint probes and 40–50 pmol of secondary oligonucleotide were added simultaneously to a 25-μl hybridization in the presence of RNase. Samples were denatured for 2.5 min at 92 °C and then allowed to hybridize overnight at 42 °C. Slides were then washed for 15 min at 60 °C in 2 × SSCT, and then for 10 min at RT in 2 × SSCT, then for 10 min at RT in 0.2 × SSC and finally mounted in Slowfade Gold mounting media with 4,6-diamidino-2-phenylindole (Invitrogen). Samples were imaged on Olympus IX-83 using a × 60, NA 1.42 lens and an Olympus XM-10 camera or a Zeiss LSM780 laser scanning confocal microscope using a × 63, NA 1.40 lens. Images processing was performed with the Olympus CellSens Dimension or Zeiss ZEN microscope-specific software as well as Adobe Photoshop. For the quantification of the number of FISH signals per nucleus, signals <1 μm apart were considered to be a single focus.

Cultures were prepared and maintained as described previously (Jaklitsch 2009 (link)). Microscopic observations were made in tap water, except where noted. Morphological analyses of microscopic characters were carried out as described earlier (Jaklitsch 2009 (link)). Methods of microscopy included stereomicroscopy using Nikon SMZ1500, Olympus SZX10 and Euromex Novex RZ 65.560, light microscopy using Euromex XHR MIC 625, Olympus BX51 and Nomarski differential interference contrast (DIC) using the compound microscopes Nikon Eclipse E600 and Zeiss Axio Imager.A1. Images and data were gathered with Nikon Coolpix 4500, Nikon DS-U2, Nikon D90, Olympus DP72 and Zeiss Axiocam 506 colour digital cameras and measured directly with the microscope, or with Olympus cellSens Dimension, NIS-Elements D v.3.0 and Zeiss ZEN Blue Edition softwares. Amyloidity of asci was assessed using Lugol or Melzer reagent. Measurements are reported as maximum and minimum in parentheses and the range representing the mean plus and minus the standard deviation of a number of measurements given in parentheses.