(A–E) Representative whole-mount tile scans (left) and high-magnification images (right) of TUBB3 (beta III tubulin)-stained calvarial defects from (B–E) d3 to d28 post-injury. (A) Uninjured control shown as a comparison. TUBB3+ nerve fibers appear green. Dashed white circles indicates margins of defect. White asterisks indicate midline suture. Upper right-hand inset depicts negative control without primary antibody application. White scale bar, 200 μm.
(F–I) Tile scan image (F) and higher magnification images of TUBB3 immunohistochemical staining of nerve fibers, including (G and I) TUBB3+ nerves at the margins of the defect and (H) center of the defect, d14 post-injury. Asterisk in tile scan indicates the midline suture. White arrowheads indicate sites of prominent re-innervation adjacent to healing bone defect edges.
(J–L) Immunohistochemical staining typifying nerve fibers within the calvarial defect at d14 post-injury, including (J) the pan-neuronal marker protein gene product 9.5 (PGP9.5), (K) the peptidergic fiber marker calcitonin gene-related peptide (CGRP), and (L) the sympathetic marker tyrosine hydroxylase (TH).
Representative images are from n = 3 mice per time point. White scale bar, 50 μm. See also Figure S2.