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Protocol publication

[…] Stacks from time-lapse recordings acquired with 0.5 s intervals were used to analyze DCV release. A 2×2 pixel region (0.4×0.4 μm) was analyzed according to the experiment as follows. Sema and NPY-pHluorin: fluorescent traces were expressed as fluorescence change (∆F) compared to initial fluorescence (F0), obtained by averaging the first four frames of the time-lapse recording. A fusion event was counted when fluorescence showed a sudden increase two standard deviations above F0. Onset of fusion was defined as the first frame with an increase of fluorescence of two standard deviations above F0. A cargo-pHl release event or punctum was scored as synaptic when the fluorescence center of such a release event/punctum was within 200 nm (±1 pixel, the approximate minimal point spread function of our system) of the Synapsin-mCherry fluorescence centroid. Extra-synaptic events were all events that did not meet this criterion. We only measured release events from neurites and excluded somatic release events. Somatic release events cannot be reliably measured using wide-field fluorescence microcopy due to the bright fluorescence from vesicles in/near the Golgi apparatus in which the intraluminal pH is not yet acidic. The total number of vesicles was automatically analyzed from the NH4+ application time lapse using SynD software (). When using NPY-mCherry: only the fusion events were scored in which NPY-mCherry fluorescence completely disappeared from a 2×2 pixel punctum after bleaching correction (ImageJ Bleaching correction plug-in). DCVs were categorized as stationary or moving based on the slope of the Kymopgraph (ImageJ, MultipleKymograph), if the slope of the line over the kymograph was different from 0 at any point of the movie, the DCV was considered moving.CAPS-1-ires-EGFP was used to rescue CAPS DKO neurons in combination with NPY-mCherry for DCV fusion assays in .Protein dispersion was analyzed by placing regions of interest (ROIs) at the synapses and analyzing the ΔF over F0 (average of the first four frames) of Synapsin-ECFP and CAPS-1-EYFP over time. ROIs not overlapping with Synapsin-ECFP were chosen for analyzing CAPS-1-EYFP dispersion at extra-synaptic sites (ΔF as above). These analyses were performed after bleaching correction (ΔF of the soma over time was used as bleaching and subtracted to the measurements). Membrane associated myristoylated EYFP was used as negative control. For co-localization analysis we used ImagJ software (National Institute of Health, USA, Plug-in JACoP). Pearson's coefficients were calculated to obtain cell wide correlation of fluorescent intensities and Mander's coefficients to obtain co-occurrence in VGLUT positive synapses, CAPS-1 puncta or NPY-puncta (). […]

Pipeline specifications

Software tools SynD, ImageJ, JACoP
Applications cryo-EM, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Calcium, Neuropeptides