(A) Dual TUNEL/α-ACTIN IHC was performed using colorimetric and fluorescent techniques.

<p>Apoptosis was barely detectable in control penile smooth muscle. Apoptosis was abundant in prostatectomy and diabetic corpora cavernosal tissue and almost all apoptosis observed co-localized with α-ACTIN in penile smooth muscle. TUNEL appears in brown (diaminobenzidine, DAB, top) and red (bottom). α-ACTIN appears in red (top) and bluish-purple (bottom). Light blue staining is unavoidable auto-fluorescence present in human penis. Arrows indicate co-localization (shown in pink on bottom). 400× magnification. (B) TUNEL/CD31 IHC shows that apoptosis is also occurring at a very low level in the endothelium. TUNEL appears in brown (DAB) and CD31 appears in red. Arrows indicate co-localization. 400× magnification. Fluorescent co-localization for TUNEL/CD31 was not possible due to the low abundance of endothelium and the high auto-fluorescence present in human penile tissue.</p

Quantification of precursor and active SHH protein by western analysis of rats that underwent bilateral CN crush and sildenafil or DMSO treatment for 7 or 14 days.

<p>Precursor and active SHH proteins were significantly increased 50% and 57% (p = 0.02 and 0.04) after 14 days of sildenafil treatment.</p

Trichrome and TUNEL were performed on corpora cavernosal tissue from control, prostatectomy, and diabetic patients.

<p>The smooth muscle/collagen ratio decreased 25% in prostatectomy and 28% in diabetic penis (p = 0.0338). The apoptotic index increased 22% in prostatectomy and 25% in diabetic penis (p = 0.0141). Arrows indicate staining. 100–400× magnification.</p

<i>In situ</i> for <i>Shh</i> RNA and IHC for SHH protein, were performed on corpora cavernosal tissue from control, prostatectomy, and diabetic patients.

<p><i>Shh</i> RNA and SHH protein are present in smooth muscle and the localization was unchanged in prostatectomy and diabetic penis. SHH protein appears decreased by visual observation in prostatectomy and diabetic penis. PTCH1 (SHH receptor) and BDNF (SHH target in the rat) were also decreased in prostatectomy and diabetic penis. Arrows indicate staining. 100–400× magnification.</p

Quantification of α-ACTIN by western analysis of corpora cavernosal tissue from control, prostatectomy and diabetic patients.

<p>α-ACTIN was significantly decreased 52% in prostatectomy and 51% in diabetic (p = 0.005) corpora cavernosa.</p

Quantification of precursor and active SHH protein by western analysis of corpora cavernosal tissue from control, prostatectomy and diabetic patients.

<p>Precursor SHH protein significantly decreased 41% in prostatectomy and 39% in diabetic patients (p = 0.0312). Active SHH protein significantly decreased 46% in prostatectomy and 48% in diabetic patients in comparison to controls (p = 0.0222). Asterisks indicate significance. Where error bars do not appear they are too small to be shown on the graph.</p

ED severity as measured by IIEF in control, prostatectomy and diabetic patients.

<p>ED severity as measured by IIEF in control, prostatectomy and diabetic patients.</p