Computational protocol: Retromer terminates the generation of cAMP by internalized PTH-receptors

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Protocol publication

[…] Spatial deconvolution of confocal images was performed using the Huygens Professional suite (Scientific Volume Imaging, Hilversum, the Netherlands). Images for deconvolution were taken according to manufacturer recommendations: Pixel size was 40 × 40 nm in X and Y dimensions, and 100 nm in the Z-axis when 3-dimensional image series were collected. Iterative deconvolution was then performed using a theoretical point spread function (PSF) based on entered values for objective NA, oil and medium refractive index, emission wavelength and estimated signal-to-noise ratio for each fluorophore.Colocalization (Pearson’s) was performed using integrated functions in Elements (Nikon) for raw confocal images. For spatially deconvolved endosomes, Pearson’s colocalization coefficient was analyzed per endosome, per time point using the JACoP plugin for ImageJ after auto-thresholding. […]

Pipeline specifications

Software tools Huygens, JACoP, ImageJ
Application Microscopic phenotype analysis
Chemicals Cyclic AMP