Eclipse te2000 u

Microscopic observation was made from fresh cell preparations by phase contrast with an Olympus BH-2 microscope at a magnification of 40x (unless otherwise stated). Pictures were obtained by a Panasonic CCD camera coupled to the microscope using DScaler software, or by a digital camera Panasonic Lumix DMC-G1K lens kit. Fluorescence microscopy images from cells treated for flow cytometry analysis were obtained from a Nikon Eclipse TE2000-U coupled with a Hamamatsu ORCA-ER CCD camera. The filters used were Nikon B-2E/C for green (GFP) and Nikon G-2A for red (PI). All pictures from each experiment were taken (gain and exposure) and processed (brightness and contrast) equally, using the Aquacosmos 1.3 program and Adobe Photoshop CS5.

For fluorescence microscopy, a fluorescence microscope NIKON ECLIPSE TE2000-U, connected to a high resolution HAMAMATSU ORCA-ER camera was used. For SEM, used to visualize protoplast cell wall regeneration, cells were fixed with 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer containing 1 M sorbitol at 4°C, overnight. Post-fixation was carried out for 2 h at room temperature with 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2). Initial dehydration was accomplished in a graded ethanol series. Then, samples were dehydrated with acetone until dried by the critical point method in liquid CO₂ (Balzers^® CPD 030). Subsequently, the specimens of the different strains were coated with graphite and gold in a vacuum evaporator (EMITECH SCD 004) and examined with a SEM JEOL Observations were carried out in JEOL JSM-6400 microscope SEM. For transmission electron microscopy (TEM), used to observe cell wall, cells were fixed in 4% paraformaldehyde, 1%, glutaraldehyde, and 0.1% PBS overnight at 4°C. Samples were incubated for 90 min in 2% osmium tetroxide and then serially dehydrated in ethanol and embebbed in EMBed-812 resin (Electron Microscopy Sciences). Thin sections (50–70 nm) were obtained by ultracut and observed in JEOL JEM 1010 TEM.