Computational protocol: Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation

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[…] Protein extracts were separated by sodium dodecyl sulfate-polacrylamide gel electrophoresis (SDS-PAGE) and transferred to onto PVDF membranes (Miliipore, Billerica, MA, USA) using a buffer continaing 25mM Tris base, pH 8.3, 192 mM glycine, 20% (vol/vol) methanol for 1h at 100 V at 4°C. Membranes were blocked for 1h in 10% milk/0.1% Tween/TBS, then incubated overnight at 4°C with primary antibodies as described in the text. After incubation in primary antibody, membranes were rinsed with 0.1% Tween/TBS three times, and subsequently incubated 2h at room temperature with secondary light-chain specific HRP-conjugated antibodies diluted 1/1000 in 10% milk/0.1% Tween/TBS. After rinsing, membrances were incubated with ECL (Thermo Scientific, Somerset, NJ) for 10 min and then revealed. For densitometry, unsaturated films were scanned using a Canon LiDE scanner (Lake Success, NY), with mean pixel density of each band measured using ImageJ software. Measurements were standardized to actin loading control, and fold change versus baseline calculated. [...] Fractal analysis was conducted on cells expressing and immunostained for the O4 antigen which identifies immature and mature OL. Image processing and analysis was done in ImageJ software using the FracLac plugin with default settings. 15 cells per well were analyzed. For proper analysis, assessed cells were isolated without contact with other cells. Imaged cells were binarized by thresholding with default settings and then thinned using the “Skeletonize” command (a thinning algorithm within ImageJ). The box-counting fractal dimension (Db) was calculated for each cell using the FracLac plugin at default settings. This value is derived from the slope of the linear regression between the log of the number of boxes occupied by pixels and the log of these boxes’ size. […]

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