Computational protocol: Rac1 plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems

Similar protocols

Protocol publication

[…] Individual spinal cords from E11.5 embryos were dissected in Hank’s balanced salt solution (HBSS), and genotyping was performed on the remaining embryonic tissue. Spinal cords were diced, washed in HBSS, and incubated in 0.05 % trypsin-EDTA for 12 min at 37 °C, washed in DMEM with 10 % heat-inactivated normal horse serum, resuspended in the same media, triturated with a fire-polished Pasteur pipette, and then filtered through a 70-μm filter. Cells were plated on sterile glass coverslips coated in poly-D-lysine (20 μg/ml) and laminin (20 μg/ml) at a density of 50,000 cells/coverslip in 24-well plates. For immunohistochemistry, cells were washed twice in PBS with 0.5 mM MgCl2 and 1 mM CaCl2 and fixed for 10 min in 4 % PFA. GFP was visualized with chicken anti-GFP (GFP-1020; Aves Lab; Tigard, OR). Axon lengths were analyzed using the NeuronJ plug-in for ImageJ. [...] The number of neurons on each cryosection was manually quantified using ImageJ software. To measure the diameter of limb motor nerves, a line perpendicular to the nerve at the designated site was drawn in Adobe Illustrator and the width of the nerve along this line was then measured. Statistical comparisons were performed in Microsoft Excel. […]

Pipeline specifications

Software tools NeuronJ, ImageJ, Adobe Illustrator
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus