Computational protocol: Torsin A Localization in the Mouse Cerebellar Synaptic Circuitry

Similar protocols

Protocol publication

[…] Cerebella were obtained from anesthetized mice at different age: P7, P14, P21, P60. Cerebella were homogenized with cold RIPA buffer (50 mM Tris pH 7.5, 300 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 MgCl2, 1 mM CaCl2, 1 mM ethylene glycol tetraacetic acid, and 1% protease inhibitor cocktail, Sigma) sonicated and left in ice for 1 hour (h); then crude lysates were centrifuged at 13000 rpm for 15 min at 4°C, the supernatant collected and quantified with Bradford assay. Protein extracts were stored at -80°C. 5 µg of each sample in Page LDS sample buffer 4x (Invitrogen) was analyzed to detect TA variation of expression during development; samples were denatured at 98°C for 5’ and were loaded onto the sodium dodecyl sulfate (SDS) polyacrylamide gel 12% (Biorad). Gels were blotted onto a 0.45 µm polyvinylidene fluoride (PVDF) membrane. Immunodetection was performed by rabbit anti-TA 1:500 (AbCam cat. num. ab34540) and mouse anti-β actin (Sigma, A5441) was used as loading control. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad), ECL-Plus reagent (GE Healthcare, Europe) and the storm 840 acquisition system were used for detection of the proteins of interest. Quantification of the bands intensity on scanned filters was achieved by ImageJ software []. The experiments were performed in triplicates, data form each experiments were normalized to adult age values and pulled together for statistical analysis. Data were statistically compared using the One-way ANOVA post hoc Tukey’s test. Differences were considered significant if p< 0.001. The results were presented as means ± SEM. [...] The quantitative colocalization analysis was performed by means of the ImageJ software, in particular by using the Just Another Colocalization Plugin (JACoP) plugin [] and the Intensity Correlation Analysis (ICA) plugin (WCIF ImageJ) as described by Li et al. (2004) []. Both analysis were conducted on synaptic structures in which both pre- and post-synaptic sites were clearly defined not only in the x-y dimension but also in the z-axis. To this aim we selected the best optical section in the z-stack in terms of intensity signal and area occupied by the synaptic structures.We first isolated sets of synaptic contacts in a single optical section and calculated the overlap coefficient r as reported by JACoP: ( ∑i (Ai x Bi ))/√(∑i (Ai)2 x ∑i(Bi)2) []; where Ai and Bi represent signal intensities of pixels for each channel. This coefficient varies from zero to one, the former corresponding to non-overlapping signals and the latter reflecting 100% of colocalization between both signals. The total number of synaptic contacts was reported for each group of synapse in the results.The ICA was performed on isolated single synaptic structures along the z-stack (0.3 µm step). To this aim, besides analyzing the best central section (as described above) we analyzed also the up and down sections. This type of analysis generates scatter plots (ICA plot) of stain A or stain B against the product of the differences from the mean (PDM) which is the product of the difference of each pixel A and B intensities from their respective means as described in Li et al. 2004 []. The resulting plots emphasize the high intensity stained pixels and allow the identification of protein pairs that vary in synchrony (positive PDM values), randomly (around 0), or independently (negative PDM values) within the cell. The ICQ value is based on the non-parametric sign-test analysis of the PDM values and is equal to the ratio of the number of positive PDM values to the total number of pixel values. The ICQ values are distributed between -0.5 and +0.5 by subtracting 0.5 from this ratio. Random staining: ICQ~0; Segregated staining: 0> ICQ > -0.5; Dependent staining: 0

Pipeline specifications

Software tools ImageJ, Coloc, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Cerebellar Diseases, Genetic Diseases, Inborn
Chemicals gamma-Aminobutyric Acid