Las af program

Slides with chromosome spreads were analysed using a fluorescence microscope, Leica DM 4000, equipped with a monochrome digital camera DFC350 FX and filter cubes corresponding to fluorochromes Alexa488, Cy3, and DAPI (Leica-Microsystems). The LAS X core computer program was used to obtain and process colour images.
Analysis of the 3D morphology of the gonads and whole-mount FISH was performed by laser scanning confocal microscopy (using a Leica TCS SP5 based on the inverted microscope Leica DMI 6000 CS, Leica-Microsystems). Nuclei were scanned in XYZ planes using lens HC PL APO 40×. Images were obtained by the LAS AF program (Leica-Microsystems, Germany).

Nocodazole was used as a MT depolymerizing drug. Vero cells were seeded and infected at an moi of 1 pfu/cell and treated with 10 µM nocodazole in DMSO 1 hour prior to infection (−1 h), at the time of infection (0 hpi), or 2 and 4 hpi (+2 and +4 hpi). To address the effect of nocodazole in endosome movement in this cell line, we detected acidic endosomes using lysotracker (75 nM), a pH-sensitive dye, for 30 min at 37 °C. Then, confocal images were taken before and after nocodazole treatment and after washing the drug and adding fresh media. Time-lapse microscopy was carried out using a Leica TCS SPE confocal microscope that included a humidified incubation chamber, a CO₂ controller and a heating unit. Selected stacks were recorded every 10 s using the Leica Microsystems LAS AF program, and the movies were displayed at 1–5 frames per second. Then, 10 µM nocodazole stopped vesicular traffic, and movement was recovered after washing, as it is a reversible drug (data not shown).

Confocal microscopy was carried out using a Leica TCS SPE confocal microscope that included a humidified incubation chamber, a CO₂ controller and a heating unit. Selected stacks were recorded every 10-s using the Leica Microsystems LAS AF program and the films were displayed at 1–5 frames/s.