Immunohistochemical staining of remnant muscle ends at the mid-bud stage.

<p>(A) Antibody staining of ColIV and MHC was used to demarcate the fibrillar muscle ends that are encircled by a ColIV-containing basal lamina (arrows). The muscle fibers taper and most are MHC^-. (B) The muscle end shows concentrated Pax7⁺ nuclei (arrows) arranged in a tract away from MHC⁺ muscle fibers and toward the blastema zone. These nuclei are larger and more ovoid than the comparatively more slender nuclei of satellite cells (asterisks) in the proximal muscle. (C) The density of Pax7⁺ nuclei in the distal muscle region is considerably higher than that in the proximal region. (D) Triple staining of Pax7 (green), MHC (green), and ColIV (red) was performed to show Pax7⁺ nuclei present in the fibrillar muscle ends. Nuclei were counterstained with Hoechst in order to differentiate between the Pax7 and MHC signals. D1, D2, and D3 are enlarged images of the respective boxed areas in D. D1´, D2´, and D3´ are images with addition of Hoechst signals in D1, D2, and D3, respectively. Red arrows indicate Pax7⁺ cells distant from the fibrillar ends and extending into the blastema. White arrows indicate Pax7⁺ cells encompassed within a ColIV-containing basal lamina. Arrowheads indicate Pax7⁺ cells partially encircled by ColIV. P→D: proximal-to-distal direction. Scale bars = 100 μm.