Computational protocol: Phosphoinositide-dependent enrichment of actin monomers in dendritic spines regulates synapse development and plasticity

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Protocol publication

[…] Data analyses were performed using ImageJ (National Institutes of Health) and NIS-Elements software. Dendritic regions of interest in hippocampal neurons were first taken as three-dimensional image stacks and then projected to two-dimensional images using the maximal intensity z-projection function from ImageJ. To analyze spine volume, we measured the integrated intensity of tdTomato or EGFP signals in all the spines selected by the Auto Thresholding function from ImageJ. Ratio images were made with a custom Ratio ImageJ Plugin after background subtraction, registration, and photobleaching correction. For FRAP assay data analysis, images were background subtracted and corrected for photobleaching. Fluorescence intensity profiles of dendritic spines were generated along 5 pixel–wide lines through the centers of dendritic spine heads of interest with the plot profile tool of ImageJ. Sholl analysis was performed by using the Anirvan Ghosh laboratory ImageJ Sholl Analysis Plugin (v1.0). Most statistical analysis was performed using SPSS Statistics (v.24; IBM Corp.).To determine the effects of exogenously expressed EGFP-actin or EGFP-actin mutants on cells, we grouped them into two groups according to the mean EGFP fluorescence intensity of their cell bodies: low-expression group < 1,500 and high-expression group > 2,500. Immunostaining of the total actin proteins using a pan-actin antibody (Ab14128; Abcam) was used to estimate the amount of overexpression of these EGFP-tagged actin proteins relative to the endogenous level. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis, FRAP
Diseases Keratitis, Dendritic, Nervous System Diseases