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).
Western Analysis of Control and CN-injured Corpora Cavernosa
Western analysis assaying for SHH protein was performed on protein samples isolated from corpora cavernosa from control (n = 5) and CN-injured (n = 5) Sprague Dawley rat penes 21 days after CN injury, as outlined previously. Proteins were separated via electrophoresis using a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad) using a Hoefer SemiPhor Semi-Dry Transfer Unit (Amersham Pharmacia, Piscataway, NJ) for 1 h. Membranes were blocked for 1 h at room temperature in 5% nonfat skim milk in PBS Tween buffer. Membranes were incubated with either 1:50 SHH (N-19, SC-1194, Santa Cruz) or 1:50000 ACTB (formerly known as p-ACTIN, Sigma) antibodies overnight at 4°C. Membranes were washed with PBS-Tween one time for 10 min and then incubated with 1:45 000 anti-goat and 1:70 000 anti-mouse secondary antibodies for 1 h at room temperature. Protein bands were visualized using HRP-conjugated anti-biotin (ECL western blotting detection reagent, Amersham) according to manufacturer’s directions and were exposed to Hyperfilm.