Figure 2.

Robust in vitro myelination in DRG explant cultures from Mtmr13-deficient mice analyzed using a semiautomated image collection method. Dissociated DRG explant cultures were generated from wild-type and Mtmr13^−/− embryos at E13.5 and cultured for 21 days under conditions that promote myelination. Myelin and axons were visualized via immunofluorescence with antibodies to myelin basic protein (MBP; magenta) and neurofilament heavy chain (NFH; green), respectively. (a and b) Low-magnification images of full SC-DRG explants from wild-type and Mtmr13^−/− mice show qualitatively similar extents of myelination. The overall shapes of explants vary considerably without respect to genotype. (c) A magnification of the indicated portion of (a), overlaid with a grid indicating the area sampled via the automated acquisition a set of 36 high-magnification images. The outermost corner of the grid was positioned at the outer edge of the myelinated explant. (d) Similar density of MBP-positive myelinated segments in SC-DRG explants from wild-type and Mtmr13^−/− mice. Myelin density: 186.6 ± 130.2 versus 267.3 ± 153.9 MBP-positive segments per mm² for wild type and Mtmr13^−/−, respectively; p = .2949; Mann–Whitney test; mean ± SD; n = 7 and 6 independent explants for wild type and Mtmr13^−/−, respectively. In assessing myelin density, an average of 1,067 myelin segments were counted per explant/coverslip (the median value was 806 myelin segments counted per explant/coverslip). (e and f) An example of a high-magnification image corresponding to one of the 36 images derived from the grid depicted in (c). Quantification of myelin segments was accomplished via analysis of images such as that shown in (e). The color white indicates the overlap of green and magenta in (f). Scale bar: 2 mm (a and b), 1.13 mm (c), 20 μm (e and f). MTMR = myotubularin-related.