Computational protocol: The Role of γ-Tubulin in Centrosomal Microtubule Organization

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Protocol publication

[…] Dual-axis electron tomography was performed as described . Briefly, 15-nm colloidal gold particles (Sigma-Aldrich) were attached to each surface of the semi-thick sections to serve as fiducial markers for subsequent image alignment. Tilt series datasets were imaged using a TECNAI F30 intermediate-voltage electron microscope (FEI) operated at 300 kV. The SerialEM program , was used to automatically acquire images every 1° over a ±60° range using a Gatan 2K×2K CCD camera at a pixel size of 1.2 nm. Tomograms from 3–4 serial sections were calculated using the IMOD software package and joined to produce a final volume containing most of the centrosome (2.4 µm×2.4 µm×0.9 µm) , . We recorded 4 wild-type centrosomes, 8 tbg-1(RNAi) centrosomes, and 7 centrosomes of spd-5(RNAi) embryos. By electron tomography we checked that GFP-tubulin labeling had no effect on the morphology of the pole-proximal microtubule ends. […]

Pipeline specifications

Software tools SerialEM, IMOD
Application cryo-electron tomography
Organisms Caenorhabditis elegans