Figure 5 of Kucerova, Mol Vis 2012; 18:139-150.


Figure 5. Expression of Hedgehog signaling pathway components in the adult corneal epithelium. A: western blot analysis of embryonic notochord/neural tissue (N) or wounded adult corneas using the 5E1 mouse anti-Shh-N monoclonal (Developmental Studies Hybridoma Bank) or R&D Systems goat anti-mShh-N polycolonal AF464. 5E1 recognizes 2 major bands at sizes predicted for Shh isoforms in embryonic tissue (left hand lane), but detects no Shh-N in the wounded adult cornea (middle lane). The goat polyclonal recognizes multiple non-Shh bands in wounded corneas. B, C: No Shh is detected by immunohistochemistry in the wild-type adult corneal epithelium by the 5E1 antibody (B), although there is strong background staining of the underlying corneal stroma by the Alexa-488 conjugated goat anti-mouse IgG1 secondary antibody (A21121; Molecular Probes), as shown in C the negative (no primary antibody) control. D: Shh localization in transverse section of E12.5 notochord and floor plate (immunohistochemistry using 5E1 monoclonal antibody) and EPtch1 expression (purple) upregulated in tissues around the notochord shown by in situ hybridization. The sense control was blank (data not shown). F: Localization of Dhh by immunohistochemistry with DAB (brown) endpoint in the wounded corneal epithelium. Wound edge is indicated by an arrow. G: No Shh in the corneal epithelium at a healing wound (arrow) edge shown by immunohistochemistry using the 5E1 monoclonal antibody. H: Upregulation of Ptch1 at the wound edge (arrow) is evidence that Hedgehog signaling is active. Scale bar: 100 μm.