Computational protocol: Impaired Function of HDAC6 Slows Down Axonal Growth and Interferes with Axon Initial Segment Development

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Protocol publication

[…] Neurons were fixed in 4% paraformaldehyde for 20 minutes and washed in PBS. For immunodetection, the coverslips were treated with 50 mM NH4Cl and incubated in blocking buffer (0.22% gelatin, 0.1% Triton X-100 in PBS) to avoid non-specific binding. The cells were then incubated for 1 hour at room temperature with the primary antibodies diluted in blocking buffer. The primary antibodies used were: mouse anti-acetylated-α-tubulin (1∶2000), mouse anti-tyrosinated-α-tubulin (1∶2000), mouse anti-MAP2 (1∶400) and mouse anti-PanNaCh (1∶75) from Sigma; mouse anti-tau-1 (1∶1000); and rabbit anti-MAP2 (1∶500) from Chemicon; mouse anti-ankyrinG (1∶100) from Invitrogen; rabbit anti-HDAC6 (1∶400) and rabbit anti-KIF5C (1∶200) from Abcam. The secondary antibodies used were a donkey anti-mouse or anti-rabbit Alexa-Fluor-488, 594 or 647 (1∶500). F-actin was stained using Alexa-Fluor-594-conjugated Phalloidin (1∶100). After staining, the coverslips were mounted with Fluoromount-G (Southern-Biotech) and the images were acquired on a vertical Axioskop-2plus microscope (Zeiss) or a confocal microscope (LSM510, Zeiss) under the same conditions to compare intensities. Differential interference contrast (DIC) images were obtained in a LSM510 confocal microscope. Analysis of axon and dendrite length and fluorescence intensities was performed with the Neuron J and ImageJ software tools. Images were prepared for presentation using the Adobe CS3 software. [...] All experiments were repeated at least three times and the results are presented as the mean and standard error of the mean (S.E.M). The number of neurons analyzed in each set of data is indicated in figure legends. Statistical differences between experimental conditions were analyzed by a t-test or paired t-test using the Sigmaplot 9.0 software. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Mitochondrial Diseases
Chemicals Sodium