Computational protocol: Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination

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Protocol publication

[…] For immunocytochemistry, coverslips were transferred to PBS, and fixed in 1% paraformaldehyde for 20 min. Cells were washed three times in PBS and then permeabilized in ice cold methanol for 20 min. Following three PBS washes, cells were blocked [5% donkey serum, 0.3% milk powder, 1% bovine serum albumin (BSA), 0.1% sodium azide, 1% dimethyl sulphoxide (DMSO)], washed in PBS, and incubated with the primary antibody overnight at 4°C. Cells were then washed with PBS, and incubated with the secondary antibody for 2 h at room temperature. The secondary antibody was washed off with PBS, and the coverslips were mounted onto Superfrost Plus microscope slides (Thermo Scientific) in Vectashield mounting medium (Vector Laboratories). Primary antibodies used and their dilations are indicated in the . Secondary antibodies used were Alexa Fluor 568, 546, 488, 405 or Pacific Blue.Myelination was quantified via confocal microscopy using systematic random sampling to ensure objective sampling across the coverslip. See for additional detail.NF200-positive surface area was taken as a measurement of axon area, while myelin basic protein (MBP) positive surface was used as a measurement of myelin area. Since measuring MBP area was a novel approach to quantify myelination, we cross-validated our results by comparing them to the most commonly used method (myelin internode count). We counted the number of internodes and quantified the myelin area in 18 separate myelinated co-culture coverslips within a single neuronal differentiation and found both set of results were highly correlated (Pearson’s R; r = 0.962, n = 18, P < 0.001; ). Hence, measurement of MBP surface area was used to quantify myelination, as it provided the more time efficient method. When comparing between conditions, the proportion of the total axonal area covered by myelin (MBP area/NF200 area) was calculated, and expressed as a fold change from control. To acquire cell numbers, DAPI positive nuclei were quantified using ‘Particle Analyser’ provided by ImageJ software. [...] Statistical analysis was performed using IBM SPSS Statistics for Mac (Version 22). Student’s t-test was used for comparison of two groups, one-way ANOVA using Tukey post hoc correction was used for more than two groups. Correlation was analysed using Pearson’s R. Results are reported as mean ± SEM. Asterisks indicate the level of significance (***P < 0.001, **P < 0.01, *P < 0.05). […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Rattus norvegicus, Homo sapiens
Chemicals Potassium, Sodium