Computational protocol: The PICK1 Ca2+ sensor modulates N-methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons

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Protocol publication

[…] Cells grown on coverslips were fixed in 4% paraformaldehyde (Thermo Fisher) in PBS (Sigma) supplemented with 2% sucrose at room temperature. Cells were permeabilized in 0.5% Nonidet P-40 for 2 min. Coverslips were blocked in 3% BSA (Sigma) for 1 h and incubated with anti-PICK1 (Abcam, ab3420; dilution, 1:100) in 3% BSA for 1 h at room temperature, followed by goat anti-rabbit Alexa Fluor 568 for 45 min (Thermo Fisher, A-11011; dilution, 1:1000) and mounted in DAPI-free medium (Sigma) onto slides. Coverslips were imaged on a Leica SP5 confocal system under a ×63/1.4 numerical aperture (NA) oil immersion or ×40/1.25 numerical aperture (NA) oil immersion objective using filters set up to image GFP, mRUBY, or Alexa Fluor 568. The Leica application suite software was used to acquire 0.37-μm stepped Z stacks throughout the depth of the cells, and maximal intensity projections generated by ImageJ Fiji are presented in this work. Image processing and co-localization analyses were performed using ImageJ and Coloc2. Three randomly selected dendrites were analyzed per neuron. Mander's fractions were measured using thresholding, values were normalized for each of at least three independent experiments, and statistical significance was determined using two-way ANOVA. All the error bars on graphs represent standard deviation of the mean. Sholl analysis was performed by thresholding the mRUBY channels and using the Sholl plugin (ImageJ Fiji) to measure the number of dendritic intersections every 10 μm over a total radius of 150 μm from the cell body. The numbers of intersections were measured from three independent neuronal cultures. Total dendritic length was measured using the Simple Neurite Tracer plugin (ImageJ Fiji) to calculate the total dendrite length of a neuron based on filling neurons with mRUBY. Traces were taken from three independent experiments, and statistical significance was determined using one-way ANOVA.Levels of PICK1 knockdown and molecular replacement were quantified by measuring integrated densities of PICK1 clusters detected using anti-PICK1 in cortical neuronal dendrites using ImageJ (Fiji). […]

Pipeline specifications

Software tools ImageJ, Coloc
Applications Laser scanning microscopy, Microscopic phenotype analysis
Chemicals N-Methylaspartate