Cooled ccd camera

Spinning disc confocal images were acquired on a Nikon TE-2000 with confocal scanner unit (Yokogawa) using a 100X NA1.45 objective using an electron multiplying CCD camera (Andor) controlled by Micro-Manager software (https://www.micro-manager.org). Epifluorescence images were acquired on a Nikon inverted microscope with a 60X NA1.4 objective illuminated by a mercury arc lamp through standard dichroic filter sets (Chroma) and collected using a cooled CCD camera (Princeton Instruments). TIR-FM images were collected on a Nikon Ti-E inverted microscope equipped for through-the-objective TIR-FM with a Apo TIRF 100X, NA1.49 objective (Nikon) with solid-state lasers of 405, 488, 561 and 647 nm (Keysight Technologies) using an EMCCD camera (Andor) controlled by NIS-Elements 4.1 software to acquire image sequences every 50 ms for 5 min. Exposure times and illumination were adjusted to remain in the dynamic range of each camera.

Immunocytochemical analysis was carried out for the detection of cell-specific markers as previously described [10 (link)]. Briefly, paraformaldehyde-fixed cells or cryostat sections were incubated in phosphate buffered saline, containing 5% normal goat serum or normal donkey serum and 0%, 0.2%, or 0.4% Triton-X100, followed by an overnight incubation in antibodies at 4°C. The list of antibodies used is given as supplementary data (Table S2). Cells were examined for epifluorescence following incubation in IgG conjugated to Cy3/FITC. Images were captured using a cooled CCD-camera (Princeton Instruments NJ, USA) and Openlab software (Improvision, MA, USA). In some cases, images were acquired using a Zeiss ApoTome Imager M2 microscope (Axiovert 200M) and captured by cooled CCD-camera (Zeiss). Axiovision 4.8 software was used for further image processing. Quantification of specific marker is presented as percentage of total DAPI positive cells. Five fields of four chambers of chamber slide or three coverslips were counted per experiment. Results were derived from three separate experiments.