Special Issue Techniques
High resolution three-dimensional imaging: Evidence for cell cycle reentry in regenerating skeletal muscle
Version of Record online: 3 JAN 2011
Copyright © 2010 Wiley-Liss, Inc.
Special Issue: Special Issue on Limb Development
Volume 240, Issue 5, pages 1233–1239, May 2011
How to Cite
Calve, S. and Simon, H.-G. (2011), High resolution three-dimensional imaging: Evidence for cell cycle reentry in regenerating skeletal muscle. Dev. Dyn., 240: 1233–1239. doi: 10.1002/dvdy.22530
- Issue online: 20 APR 2011
- Version of Record online: 3 JAN 2011
- Manuscript Accepted: 24 NOV 2010
- DARPA. Grant Number: Restorative Injury Repair BAA04-12 Addendum B; Grant sponsor: The Chicago Community Trust
Additional Supporting Information may be found in the online version of this article.
|DVDY_22530_sm_Suppfig1.tif||1429K||Supporting Figure 1 Probe penetration is dependent on time but not imbedding medium. Incubation of both primary and secondary probes for 1 day at 4°C was sufficient to label the entire diameter of myofibers. Forelimbs at 19 days postamputation (dpa) were imbedded in either sucrose-agarose (S-A) or OCT, sectioned and stained with primary antibodies (1 day or 2 days at 4°C) then secondary probes (2 hr or 4 hr at room temperature or 1 day at 4°C). X-Z orthogonal sections of confocal stacks, taken at ×25, were used for comparison of probe penetration. Phalloidin = red, laminin = green, tenascin-C = white, DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) = blue. Scale bar = 25 μm.|
|DVDY_22530_sm_Suppfig2.tif||462K||Supporting Figure 2 Supp. Fig. S2. Permeabilization of thick sections substantially enhances probe penetration. Sections of 19 days postamputation forelimbs were fixed with 4% paraformaldehyde then incubated in 0.5% Triton X-100 for 0 to 60 min before staining. Phalloidin = red, laminin = green, tenascin-C = white, DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) = blue. Scale bar = 25 μm.|
|DVDY_22530_sm_Suppfig3.tif||4051K||Supporting Figure 3 Additional evidence from a different muscle fiber demonstrating myonuclear EdU incorporation. A: Three-dimensional (3D) rendering of EdU+ nuclei (white) within skeletal muscle (red) of a 7 days postamputation regenerating forelimb. A mononuclear mesodermal cell that reentered the cell cycle (arrowhead) is separated from the myofiber by the basement membrane (laminin, green); moreover, an EdU+ syncytial nucleus is also present (arrow indicating absence of laminin envelope). B,C: Two-dimensional orthogonal views of a peripherally located EdU+ myonucleus. DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) = blue. Compilation of 18 z-slices rendered in 3D using ImagePro Analyzer. Scale bars = 20 μm. ×25.|
|DVDY_22530_sm_Suppfig4.tif||2191K||Supporting Figure 4 EdU+ nuclei within myofiber syncytium (arrows) originate from fusion of myoblasts. Nuclei appear to be centrally located, potentially an indication of newly regenerated myofibers. Phalloidin = red, laminin = green, tenascin-C = white, DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) = blue. Scale bar = 25 μm. ×25.|
|DVDY_22530_sm_Suppmovie1.mov||3350K||Supporting movie 1 Three-dimensional rendering of newt myofiber in situ. Phalloidin = red, laminin = green, DAPI = blue. ×25. See Supp. Fig. S2.|
|DVDY_22530_sm_Suppmovie2.mov||9902K||Supporting movie 2 Three-dimensional rendering of myonuclear cell cycle reentry. Phalloidin = red, laminin = green, EdU = white, DAPI = blue. ×25. See Figures 3 and 4.|
|DVDY_22530_sm_Supptable1.doc||31K||Supporting Table 1 Primary detection reagents used for immunohistochemistry.|
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