Bands were quantified by densitometry using Kodak 1D software (Rochester, NY). Quantification of bands was performed by determining the ratio of the density of SHH divided by ACTB to eliminate differences in protein loading. Significant differences in protein abundance of SHH/ACTB were determined in control and CN-injured penes using a t-test (Microsoft’s Excel program). Samples were run five times, and the ratios for each sample were averaged.
Immunohistochemical analysis (IHC) was performed as previously outlined on adult Sprague Dawley rats (P120, n = 6) assayed for goat polyclonal SHH (N-19, SC-1194, Santa Cruz), SHH (C-18, SC-1195, Santa Cruz), and PTCH1 (G-19, SC-6149, Santa Cruz) using a 1/100 dilution of primary antibody. Dual staining was performed using SHH (N-19)/ PECAM1 (formerly known as CD31, mouse monoclonal antibody, Chemicon International), SHH (N-19)/ACTA1 (formerly known as a-ACTIN, mouse monoclonal antibody, Sigma-Aldrich), SHH(N-19)/prolyl 4-hydroxylase (P4HB, mouse antibody, ACRIS Antibodies), PTCH1 (G-19)/PECAM1 (mouse monoclonal antibody, Chemicon International), and PTCH1 (G-19)/ ACTA1 (mouse monoclonal antibody, Sigma-Aldrich) using a 1/100 dilution of primary antibodies.