E800 phase contrast fluorescence microscope

Generally, cells were mounted on glass slides or agarose pads and observed under a Nikon E800 phase contrast/fluorescence microscope equipped with Texas Red and FITC filter sets and ×100 and ×60 oil objective lenses. Some microscopy was done with a Zeiss Axio Imager Z2 epifluorescence microscope equipped with a Hamamatsu Orca-Flash4.0 sCMOS camera, 46HE and 63HE filter sets, and a ×100 oil objective lens. Time-lapse microscopy was done with live cells mounted on an agarose pad using the Zeiss microscope equipped with automated autofocus. Time series were processed with ZEN imaging software (Zeiss). FRAP assays were performed with confocal illumination provided by an LMM5 laser launch (Spectral Applied Research). Confocal images were acquired on an Olympus IX-81 microscope equipped with a ×100 oil objective lens, a Yokogawa spinning disk CSU-X1, and an ORCA-Flash 4.0 sCMOS camera. The supply and integration of all imaging components were carried out by Biovision Technologies.

Images of stimulation experiments were acquired using a Nikon E800 phase contrast/fluorescence microscope equipped with a 10× lens objective. Fluorescence microscopy of transfer assays was done with a 60× oil lens objective and fluorescein isothiocyanate (FITC) or Texas Red filter sets.