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[…] For fluorescence microscopy cells were grown on coverslips and fixed in PTEMF buffer (20 mM PIPES, pH 6.8, 0.2% Triton X-100, 10 mM EGTA, 1 mM MgCl2, 4% formaldehyde) or methanol at -20°C (for CENP-A pS7), respectively. Images of randomly selected cell were acquired as z-stacks using a DeltaVision microscope (GE Healthcare) on an Olympus IX71 base (Applied Precision, WA, USA), equipped with a Plan Apochromat N 60x/NA1.42 oil immersion objective (Olympus) and a CoolSNAP HQ2 camera (Photometrics). Serial optical sections were deconvolved and projected using SoftWorx software (GE Healthcare). Images were quantified as previously described () using automated pipelines run by Cell Profiler software (). Results from 2–3 independent experiments were pooled and statistical analysis was done with GraphPad Prism software. Error bars on histograms illustrate SEM. Scale bars represent 10 μm.For time-lapse imaging, cells were imaged using a Nikon ECLIPSE Ti microscope equipped with a CoolLED pE-1 illumination system and a 20x/NA0.75 air Plan Apochromat objective (Nikon) in a climate-controlled environment. Images were acquired at multiple positions at indicated time intervals. MetaMorph 7.7 software (MDS Analytical Technologies, Sunnyvale, CA, USA) was used for acquisition and processing of data. FRET, FRAP, and high sensitivity microscopy (monitoring endogenously EGFP-tagged proteins) experiments were carried out using a spinning disk confocal system (Intelligent Imaging Innovations) based on a Zeiss Axio Observer stand equipped with a Photometric Evolve 512 back-illuminated EMCCD camera, 63x/NA1.4 plan apochromat objective and diode lasers and run by SlideBook software. FRET analyses were carried out by excitation with a 440 nm diode laser and by recording of CFP (CFP signal) and YFP (FRET signal) fluorescence emission in z-stacks. Background-corrected FRET ratios (CFP signal/FRET signal) were calculated in ImageJ using the Ratio Plus plugin. FRAP analysis of EGFP-Bub1 was performed with a 488 diode laser on one KT pair per cell. Overall bleaching was corrected using the signal intensities at a cytoplasmic region not targeted for photobleaching (average of the first 4 frames). Fluorescence recovery half-times and plateaus were determined by non-linear curve fitting based on a one-phase association in Prism software (GraphPad). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications SPIM, Microscopic phenotype analysis
Chemicals Paclitaxel