This was insufficient time for complete recovery, so an additional time point (6 wk after bead injection) was added to ensure complete recovery to normal morphology after SHH inhibition. Penes were harvested, fixed in 4% paraformaldehyde, and embedded in paraffin for sectioning. IHC was performed on control and SHH inhibitor-treated penes assaying for SHH (N-19, SC-1194, Santa Cruz) and ACTA1 (Sigma, St. Louis, MO) proteins as described above. Bead technology has previously been used successfully for delivery of proteins and antibodies to target tissues.
SHH Protein Injection at the Time of CN Injury
Affi-Gel beads (100-200 mesh, Bio-Rad Laboratories, Hercules, CA) were equilibrated with either 1X recombinant mouse SHH peptide (n = 10, 7.5 ig per animal, R & D Systems), 2X SHH peptide (n = 9, 15 ig per animal, R & D Systems), or PBS (control, n = 9) overnight at 4°C. Approximately 30-40 beads were injected directly into the corpora cavernosa of control and SHH peptide-treated P120 rat penes at the time of bilateral CN injury surgery (described above).