Thin sections were cut and stained with 2% uranyl acetate and 3% lead citrate. Electron microscopy was performed using a JEOL 100CX Transmission Electron Microscope to identify in which cell type apoptosis was taking place. Apoptosis was identified by the presence of condensed chromatin, nuclear fragmentation, and cytoplasmic blebbing, which are common in cells undergoing apoptosis.
Reversibility of SHH Inhibition
Affi-Gel beads (100-200 mesh, Bio-Rad Laboratories, Hercules, CA) were equilibrated with 5E1 SHH inhibitor (n = 3 at each time postinjection, 1-3 ig/ ml, Jessel, Hybridoma bank at the University of Iowa), or PBS (control, n = 2 at each time postinjection) overnight at 4°C. Approximately 30-40 beads were injected directly into the corpora cavernosa of P120 Sprague Dawley rat penes. Rats used for these experiments were normal rats that had not undergone CN injury. Rats were killed 10 days, 4 wk, and 6 wk following Affi-gel bead injection (0, 2, and 4 wk after SHH inhibitor in the beads was depleted). Four weeks after bead injection was chosen as a time point to examine if SHH inhibition was reversible, since it represented a comparable amount of time for the tissue to recover as it was exposed to SHH inhibitor.