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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22530_sm_Suppfig1.tif1429KSupporting 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.tif462KSupporting 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.tif4051KSupporting 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.tif2191KSupporting 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.mov3350KSupporting 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.mov9902KSupporting 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.doc31KSupporting Table 1 Primary detection reagents used for immunohistochemistry.

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