Computational protocol: Requirement of cAMP Signaling for Schwann Cell Differentiation Restricts the Onset of Myelination

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Protocol publication

[…] Cells were fixed with 4% paraformaldehyde followed by -20°C methanol. Cultures were blocked in 5% normal goat serum in PBS, incubated overnight at 4°C with the appropriate dilution of the primary antibody and then rinsed three times with PBS prior to incubation with Alexa-conjugated secondary antibodies. Labeling of cell surface O1 antigens was done by incubating living cells with hybridoma culture supernatant (20 min, RT) before paraformaldehyde fixation. An additional permeabilization step with a solution of 95% ethanol, 5% acetic acid was included for optimal detection of MBP. Cells were mounted with Vectashield containing DAPI (Vector Labs, Burlingame, CA) and analyzed by conventional or automated fluorescence microscopy using 10x to 40x magnification objectives. Conventional microscopy images were taken using a cooled digital black and white CCD camera (SensiCam QE, Cooke Corp.) coupled to an Olympus IX70 inverted fluorescence microscope. Images (8 bit, tiff format) were artificially colorized in RGB format, digitally processed and arranged for presentation using Adobe Photoshop V7.0 and Adobe Illustrator CS3. For cell quantification analysis, pictures from random fields were taken at 10–20x magnification and the number of cells labeled positive for the indicated markers was determined in reference to the total number of cells (DAPI staining). Cells were classified as positive or negative for the expression of Krox-20 (nuclear localization), O1 (membrane localization) and MBP (myelin segments) in comparison to non-treated controls and without regard to the variability of staining intensity shown by individual cells. At least 2,000 cells were analyzed in each sample. [...] The statistical significance of the different treatments was analyzed relative to each control group by a one-way analysis of variance (ANOVA) follow by a post-hoc Tukey test using IBM SPSS statistics software. A p-value <0.05 was interpreted as statistically significant. Data from a representative experiment out of at least three independent experiments performed are shown. All experimental conditions were analyzed in triplicate samples. […]

Pipeline specifications

Software tools Adobe Illustrator, SPSS
Applications Miscellaneous, Conventional fluorescence microscopy
Chemicals Colforsin