Computational protocol: siRNA screen of ES cell-derived motor neurons identifies novel regulators of tetanus toxin and neurotrophin receptor trafficking

Similar protocols

Protocol publication

[…] Quantification of western blots and immunofluorescence staining was performed using ImageJ for all experiments except the siRNA screen and the validation re-screen. Typically, the fluorescence staining of the area of interest was thresholded and the number of thresholded voxels measured. Immunostaining for neuronal-specific proteins, such as βIII tubulin, was used for normalization purposes.For the siRNA screen, image quantification of fluorescent HCT and αp75NTR was performed using Cell Profiler ( RGB images were split and the green GFP channel (motor neuron reporter) was used to create a cell mask, which was then subtracted from the red AlexaFluor555-HCT and blue AlexaFluor647-αp75NTR channels (Figure ). Individual HCT and p75NTR-positive organelles were then identified in each motor neuron and quantified. […]

Pipeline specifications

Software tools ImageJ, CellProfiler
Application Microscopic phenotype analysis
Organisms Mus musculus