Computational protocol: Cellular and Molecular Characterization of Multipolar Map5-Expressing Cells: A Subset of Newly Generated, Stage-Specific Parenchymal Cells in the Mammalian Central Nervous System

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Protocol publication

[…] All images were collected with the confocal microscope. Images were processed using Adobe Photoshop CS4 (Adobe Systems, San Jose, CA). Only general adjustments to color, contrast, and brightness were made.Quantitative analysis (cell densities, marker coexpression, soma diameters and cell process lenght) were performed by means of the Neurolucida software (Micro-Brightfield, Colchester,VT), Imaris 7.4.2 software (Bitplane AG, Zurich, Switzerland) or by confocal analysis. Countings has been conducted on 3 to 5 representative coronal (brain) or sagittal (cerebellum) cryostat sections, from each animal. Three animals were analyzed for each time point or experimental condition. For cell densities: the areas of the regions of interest were determined per section using a suitably calibrated Neurolucida-microscope setup. Data are expressed as densities (cell/mm2) and presented as means ± SEM. For multiple/double staining, the number of inspected cells ranged from 50 to 300 in total / 3 animals (see also S2). For Ki67 and BrdU staining, at least 50 cells in total / 3 animals were analyzed (see also S2). For morphometric analysis (soma diameters, total process lenght), at least 30 cells were randomly chosen in each area analyzed in three animals. We analyzed three different CNS areas: cerebral cortex, corpus callosum and cerebellar cortex. The soma diameter was calculated for each cell by measuring its minimum (min) and maximum extent (max) in two orthogonal directions (). Average soma diameter was obtained averaging the two values. The total length of mMap5 cell processes was calculated on confocal images processed with Imaris 7.4.2 software, using the “Autopath” filament function, to trace labeled processes. All morphometric data were presented as means± SEM.Statistical analysis was carried out by the Statistical Package for the Social Science 14.0 (SPSS, Chicago, IL) and included two way ANOVA test followed by Bonferroni's post hoc analysis (to compare mean values) and Student's t test (to compare two groups). P<0.05 was considered statistically significant. […]

Pipeline specifications

Software tools Neurolucida, Imaris
Application Laser scanning microscopy
Organisms Mus musculus, Oryctolagus cuniculus, Homo sapiens