Application suite 3d deconvolution software

Immunofluorescence microscopy was carried out as described previously (Torres et al., 2010 (link)). HeLa or HEK293 stable cell lines were induced to express Mid1 or Mid2 for 24 hours and fixed, permeabilized, and co-stained with the indicated antibodies. Images were captured with a Leica DMI6000 microscope (Leica DFC360 FX Camera, 63×/1.40-0.60 NA oil objective, Leica AF6000 software). Images were deconvolved with Leica Application Suite 3D Deconvolution software and exported as TIFF files. For quantifying spindle and cytokinetic defects, 100 cells from 3 independent experiments were counted and the data are presented as the average ± SD. For time-lapse microscopy, HeLa cells were transfected with indicated siRNAs for 24 hours, arrested in G1/S with 2 mM thymidine for 18 hours, washed, and released. Cells were imaged live 5 hours post release for 20 hours using a Leica DMI6000 microscope, described above, at 37 °C, and 5% CO₂ using a 20×/0.4 NA air objective. Images were converted to AVI movies. Each frame represents a five-minute interval. For details and a list of antibodies used, see Supplemental Experimental Procedures.