Actin Cytoskeleton Visualization in PC12 Cells

PC12 cells were fixed in a solution consisting of 1x PEM (80 mM PIPES, 5 mM EGTA, and 1 mM MgCl₂, pH 6.8) and 0.3% glutaraldehyde. Cells were permeablized by the addition of 0.05% Triton X-100 (Sigma Aldrich) [36 (link)]. Sodium borohydride (2 mg/ml) was used for glutaraldehyde quenching. Cells were labeled with rhodamine phalloidin (Cytoskeleton, Denver CO, USA) in order to visualize F-actin and the structural contour. Images were captured using an inverted Zeiss LSM800 confocal microscope and with the Zen software package (Carl Zeiss AG, Oberkochen, Germany). Morphometric measures were carried out in ImageJ (NIH, Bethesda, MD, USA). Neurites longer than the soma (>10 μm) were reconstructed using the Vaa3D software [37 (link),38 (link)] (n = 20 reconstructions per condition). All reconstructed SWC files were deposited in the public repository, Neuromorpho.org an open source database for neuronal reconstructions. All raw data measurements for cell growth of both WT and α7_345-348A expressing PC12 cells are provided in S2 File. Growth cones were identified based on the criteria described in [39 (link)].