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FM1-43 was then secreted by application of 90 mM K+, followed by a 3 min wash with Tyrode's buffer and the previously acquired fields were imaged again under the exactly the same imaging conditions (i. e. illumination setting, exposure time and binning); this defined the 'release' set of images.
For immunofluorescence staining, including Figs 1i, 2g,f, 3a, 5a,e,h and Supplementary Figs 1h, 2a,b,d, 5c,f,h, 6a,b, at least 2 different HPCa samples were used, and multiple fields were imaged on each slide.
10 15 unique fields were imaged for each experiment.
The same microscopic fields were imaged before and after treatment for contraction analysis.
For every cover slip 5 9 random fields were imaged with a Biorad Confocal Microscope MRC1024 using a 10× objective.
Random fields were imaged under constant conditions using a DMI4000B microscope (Leica micosystems) using a 60× objective.
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For each individual well of the 96-well plates, half of the field was imaged.
The same field was imaged from 30 min to 4 h.
The field was imaged before, 10 min, and 30 min after saline application (control), and changes in puncta were analyzed similarly to puncta of synaptophysin-GFP.
A 10× field was imaged using time-lapse microscopy.
Every field was imaged as a Z-stack spanning all the beads contained in it.
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