Secondary antibodies used were Alexa Fluor 488 rabbit anti-goat (1/300, Molecular Probes) and Alexa Fluor 594 chicken antimouse (1/300, Molecular Probes). Negative controls were performed with secondary only (without primary) to test for nonspecific staining and autofluorescence. Sections were mounted using Pro-Tex Mounting Medium (Baxter Diagnostics, Inc., Pittsburgh, PA). Microscopy was performed using a dual light and fluorescent microscope (Leitz) and photographed using a Nikon digital camera.
Control (n = 6) and CN-injured (n = 6) penes 21 days after nerve injury were assayed with SHH (N-19, Santa Cruz) and PTCH1 (G-19, Santa Cruz) as described above.
TUNEL Assay for Apoptosis
Staining for apoptosis was performed on control (n = 4 at each time point) and CN-injured (n = 4 at each time point) penes 2, 4, 7, and 21 days after CN injury (CN21, respectively), using the ApopTag kit (Intergen, Purchase, NY). Fluorescent apoptotic cells were observed under a fluorescent microscope (Leitz) and photographed using a Nikon digital camera.
Electron microscopy was performed as described previously. Control (n = 4) and CN-injured (n = 4) penes 21 days after CN injury were fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, dehydrated, and embedded in Epon resin.