Computational protocol: Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures

Similar protocols

Protocol publication

[…] Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.3% Triton X-100 in PBS. Cells were then blocked with blocking solution (2% skim milk, 0.1% Tween20 in PBS) and incubated with the primary antibodies at 4°C overnight. After washing with PBS, cells were incubated with secondary antibodies at 4°C overnight and stained with DAPI at room temperature for 10 minutes. Morphologies of immunostained cells were analyzed by a laser-scanning confocal microscope FV1000 (Olympus) with a 20× dry objective (N.A. 0.75, Olympus), 40× dry objective (N.A. 0.95) and a 100× oil-immersion objective (N.A. 1.4). Detailed methods for immunostaining and confocal analyses of labeled neurons were described previously [].Axon/dendrite specification was identified by immunostaining with anti-Ankyrin G and the morphological criteria described previously [, ]. Cells that were clearly free from other transfected cells were selected and analyzed by virtue of soluble GFP or TdTomato fill. Dendrites were traced with the aid of Neurolucida software (MBF Bioscience) and processed for quantitative analysis using Neurolucida Explore (MBF Bioscience) and ImageJ (NIH). The principal dendrite was defined as the longest dendrite of a neuron. To analyze the localization of the Golgi apparatus, the nucleus center of mass was set as a polar coordinate origin, and the direction to the base of the principal dendrite was defined as φ = 0. A neuron was separated into three regions by φ interval: (–π/4, π/4] as the apical region, (π/4, 3π/4] and (-3π/4, -π/4] as the lateral region, and the remaining interval as the basal region. The area occupied with the GM130 signal in each region relative to the whole GM130 area in the soma was calculated. All data are expressed as mean ± SEM. […]

Pipeline specifications

Software tools Neurolucida, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Keratitis, Dendritic