Effect of SHH Inhibition and Supplementation on VEGFA Signaling in Intact Rats
Affi-Gel beads (100-200 mesh, Bio-Rad Laboratories, Hercules, CA) were equilibrated with either 5E1 SHH inhibitor (n = 5, 1-3 ig/ml, Jessel, Hybridoma bank at the University of Iowa), recombinant mouse SHH peptide (n = 5, 7.5 ig per animal, R & D Systems), or mouse IgG (control, n = 5, 3 ig/ml) overnight at 4°C. Approximately 30^0 beads were injected directly into the corpora cavernosa of P120 rat penes. Sprague Dawley rats used for these experiments were normal rats that had not undergone CN injury.
Rats were killed 7 days postinjection. Penes were harvested and fixed in 4% paraformaldehyde for sectioning. IHC was performed on control, SHH protein, and SHH inhibitor-treated penes assaying for vascular endothelial growth factor (VEGFA, goat polyclonal antibody, Santa Cruz, 200 ig/ml and mouse monoclonal antibody, Chemicon International) as described above. Sections were stained with diaminobenzidine (DAB) and mounted using Pro-Tex Mounting Medium (Baxter Diagnostics, Inc., Pittsburgh, PA). TUNEL staining for apoptosis was also performed on control, SHH-inhibited, and SHH protein-treated penes as described above.